Chromone Inhibitors of S-Nitrosoglutathione Reductase

ABSTRACT

The present invention is directed to inhibitors of S-nitrosoglutathione reductase (GSNOR), pharmaceutical compositions comprising such GSNOR inhibitors, and methods of making and using the same.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. application Ser. No.14/168,595, filed Jan. 30, 2014. U.S. application Ser. No. 14/168,595 isa continuation of U.S. application Ser. No. 13/912,287, filed Jun. 7,2013, now U.S. Pat. No. 8,669,381. U.S. application Ser. No. 13/912,287is a divisional of U.S. application Ser. No. 13/521,820, filed Jul. 12,2012, now U.S. Pat. No. 8,481,590, issued Jul. 9, 2013. U.S. applicationSer. No. 13/521,820 is a 35 U.S.C. §371 national phase application ofPCT/US2010/024035, filed Feb. 12, 2010 (WO 2011/099978). Each of thereferenced applications is incorporated herein by reference in itsentirety.

FIELD OF THE INVENTION

The present invention is directed to novel chromone inhibitors ofS-nitrosoglutathione reductase, pharmaceutical compositions comprisingsuch inhibitors, and methods of making and using the same.

BACKGROUND OF THE INVENTION

The chemical compound nitric oxide is a gas with the chemical formulaNO. NO is one of the few gaseous signaling molecules known in biologicalsystems, and plays an important role in controlling various biologicalevents. For example, the endothelium uses NO to signal surroundingsmooth muscle in the walls of arterioles to relax, resulting invasodilation and increased blood flow to hypoxic tissues. NO is alsoinvolved in regulating smooth muscle proliferation, platelet function,neurotransmission, and plays a role in host defense. Although nitricoxide is highly reactive and has a lifetime of a few seconds, it canboth diffuse freely across membranes and bind to many molecular targets.These attributes make NO an ideal signaling molecule capable ofcontrolling biological events between adjacent cells and within cells.

NO is a free radical gas, which makes it reactive and unstable, thus NOis short lived in vivo, having a half life of 3-5 seconds underphysiologic conditions. In the presence of oxygen, NO can combine withthiols to generate a biologically important class of stable NO adductscalled S-nitrosothiols (SNO's). This stable pool of NO has beenpostulated to act as a source of bioactive NO and as such appears to becritically important in health and disease, given the centrality of NOin cellular homeostasis (Stamler et al., Proc. Natl. Acad. Sci. USA,89:7674-7677 (1992)). Protein SNO's play broad roles in cardiovascular,respiratory, metabolic, gastrointestinal, immune and central nervoussystem function (Foster et al., 2003, Trends in Molecular MedicineVolume 9, Issue 4, April 2003, pages 160-168). One of the most studiedSNO's in biological systems is S-nitrosoglutathione (GSNO) (Gaston etal., Proc. Natl. Acad. Sci. USA 90:10957-10961 (1993)), an emerging keyregulator in NO signaling since it is an efficient trans-nitrosatingagent and appears to maintain an equilibrium with other S-nitrosatedproteins (Liu et al., 2001) within cells. Given this pivotal position inthe NO—SNO continuum, GSNO provides a therapeutically promising targetto consider when NO modulation is pharmacologically warranted.

In light of this understanding of GSNO as a key regulator of NOhomeostasis and cellular SNO levels, studies have focused on examiningendogenous production of GSNO and SNO proteins, which occurs downstreamfrom the production of the NO radical by the nitric oxide synthetase(NOS) enzymes. More recently there has been an increasing understandingof enzymatic catabolism of GSNO which has an important role in governingavailable concentrations of GSNO and consequently available NO andSNO's.

Central to this understanding of GSNO catabolism, researchers haverecently identified a highly conserved S-nitrosoglutathione reductase(GSNOR) (Jensen et al., Biochem J., 331:659-668 (1998); Liu et al.,Nature, 410:490-494 (2001)). GSNOR is also known asglutathione-dependent formaldehyde dehydrogenase (GS-FDH), alcoholdehydrogenase 3 (ADH class 3) (Uotila and Koivusalo, Coenzymes andCofactors., D. Dolphin, ed. pp. 517-551 (New York, John Wiley & Sons,1989)), and alcohol dehydrogenase 5 (ADHS) Importantly GSNOR showsgreater activity toward GSNO than other substrates (Jensen et al., 1998;Liu et al., 2001) and appears to mediate important protein and peptidedenitrosating activity in bacteria, plants, and animals. GSNOR appearsto be the major GSNO-metabolizing enzyme in eukaryotes (Liu et al.,2001). Thus, GSNO can accumulate in biological compartments where GSNORactivity is low or absent (e.g. airway lining fluid) (Gaston et al.,1993).

Yeast deficient in GSNOR accumulate S-nitrosylated proteins which arenot substrates of the enzyme, which is strongly suggestive that GSNOexists in equilibrium with SNO-proteins (Liu et al., 2001). Preciseenzymatic control over ambient levels of GSNO and thus SNO-proteinsraises the possibility that GSNO/GSNOR may play roles across a host ofphysiological and pathological functions including protection againstnitrosative stress wherein NO is produced in excess of physiologicneeds. Indeed, GSNO specifically has been implicated in physiologicprocesses ranging from the drive to breathe (Lipton et al., Nature,413:171-174 (2001)) to regulation of the cystic fibrosis transmembraneregulator (Zaman et al., Biochem Biophys Res Commun, 284:65-70 (2001),to regulation of vascular tone, thrombosis and platelet function (deBelder et al., Cardiovasc Res. 1994 May; 28(5):691-4. (1994); Z.Kaposzta, A et al., Circulation; 106(24): 3057-3062, 2002) as well ashost defense (de Jesus-Berrios et al., Curr. Biol., 13:1963-1968(2003)). Other studies have found that GSNOR protects yeast cellsagainst nitrosative stress both in vitro (Liu et al., 2001) and in vivo(de Jesus-Berrios et al., 2003).

Collectively data suggest GSNO as a primary physiological ligand for theenzyme S-nitrosoglutathione reductase (GSNOR), which catabolizes GSNOand consequently reduces available SNO's and NO in biological systems(Liu et al., 2001), (Liu et al., Cell, (2004), 116(4), 617-628), and(Que et al., Science, 2005, 308, (5728):1618-1621). As such, this enzymeplays a central role in regulating local and systemic bioactive NO.Since perturbations in NO bioavailability has been linked to thepathogenesis of numerous disease states, including hypertension,atherosclerosis, thrombosis, asthma, gastrointestinal disorders,inflammation and cancer, agents that regulate GSNOR activity arecandidate therapeutic agents for treating diseases associated withnitric oxide imbalance.

Currently, there is a great need in the art for diagnostics,prophylaxis, ameliorations, and treatments for medical conditionsrelating to increased NO synthesis and/or increased NO bioactivity. Inaddition, there is a significant need for novel compounds, compositionsand methods for preventing, ameliorating, or reversing otherNO-associated disorders. The present invention satisfies these needs.

SUMMARY OF THE INVENTION

The present invention provides novel chromone compounds useful asS-nitrosoglutathione reductase (“GSNOR”) inhibitors. The inventionencompasses pharmaceutically acceptable salts, prodrugs, and metabolitesof the described GSNOR inhibitors. Also encompassed by the invention arepharmaceutical compositions comprising at least one GSNOR inhibitor andat least one pharmaceutically acceptable carrier.

The compositions of the present invention can be prepared in anysuitable pharmaceutically acceptable dosage form.

The present invention provides a method for inhibitingS-nitrosoglutathione reductase in a subject in need thereof. Such amethod comprises administering a therapeutically effective amount of apharmaceutical composition comprising at least one GSNOR inhibitor or apharmaceutically acceptable salt thereof, a prodrug or metabolitethereof, in combination with at least one pharmaceutically acceptablecarrier. The GSNOR inhibitor can be a novel compound according to theinvention, or it can be a known compound which previously was not knownto be an inhibitor of GSNOR.

The present invention also provides a method of treating a disorderameliorated by NO donor therapy in a subject in need thereof. Such amethod comprises administering a therapeutically effective amount of apharmaceutical composition comprising at least one GSNOR inhibitor or apharmaceutically acceptable salt thereof, a prodrug, or metabolitethereof, in combination with at least one pharmaceutically acceptablecarrier. The GSNOR inhibitor can be a novel compound according to theinvention, or it can be a known compound which previously was not knownto be an inhibitor of GSNOR.

The present invention also provides a method of treating a cellproliferative disorder in a subject in need thereof. Such a methodcomprises administering a therapeutically effective amount of apharmaceutical composition comprising at least one GSNOR inhibitor or apharmaceutically acceptable salt thereof, a prodrug, or metabolitethereof, in combination with at least one pharmaceutically acceptablecarrier. The GSNOR inhibitor can be a novel compound according to theinvention, or it can be a known compound which previously was not knownto be an inhibitor of GSNOR.

The methods of the invention encompass administration with one or moresecondary active agents. Such administration can be sequential or in acombination composition.

Although methods and materials similar or equivalent to those describedherein can be used in the practice or testing of the present invention,suitable methods and materials are described below. All publiclyavailable publications, patent applications, patents, and otherreferences mentioned herein are incorporated by reference in theirentirety. In the case of conflict, the present specification, includingdefinitions, will control.

Both the foregoing summary and the following detailed description areexemplary and explanatory and are intended to provide further details ofthe compositions and methods as claimed. Other objects, advantages, andnovel features will be readily apparent to those skilled in the art fromthe following detailed description.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS A. Overview of theInvention

Until recently, S-nitrosoglutathione reductase (GSNOR) was known tooxidize the formaldehyde glutathione adduct, S-hydroxymethylglutathione.GSNOR has since been identified in a variety of bacteria, yeasts, plantsand animals and is well conserved. The proteins from E. coli, S.cerevisiae and mouse macrophages share over 60% amino acid sequenceidentity. GSNOR activity (i.e., decomposition of S-nitrosoglutathionewhen NADH is present as a required cofactor) has been detected in E.coli, in mouse macrophages, in mouse endothelial cells, in mouse smoothmuscle cells, in yeasts, and in human HeLa, epithelial and monocytecells. Human GSNOR nucleotide and amino acid sequence information can beobtained from the National Center for Biotechnology Information (NCBI)databases under Accession Nos. M29872, NM_(—)000671. Mouse GSNORnucleotide and amino acid sequence information can be obtained from NCBIdatabases under Accession Nos. NM_(—)007410. In the nucleotide sequence,the start site and stop site are underlined. CDS designates codingsequence. SNP designates single nucleotide polymorphism. Other relatedGSNOR nucleotide and amino acid sequences, including those of otherspecies, can be found in U.S. Patent Application 2005/0014697.

In accord with the present invention, GSNOR has been shown to functionin vivo and in vitro to metabolize S-nitrosoglutathione (GSNO) andprotein S-nitrosothiols (SNOs) to modulate NO bioactivity, bycontrolling the intracellular levels of low mass NO donor compounds andpreventing protein nitrosylation from reaching toxic levels.

Based on this, it follows that inhibition of this enzyme potentiatesbioactivity in all diseases in which NO donor therapy is indicated,inhibits the proliferation of pathologically proliferating cells, andincreases NO bioactivity in diseases where this is beneficial.

B. S-Nitrosoglutathione Reductase Inhibitors 1. Inventive Compounds

The present invention provides pharmaceutical agents that are potentinhibitors of GSNOR. In particular, provided are substituted chromoneanalogs that are inhibitors of GSNOR having the structure depicted below(Formula I), or a pharmaceutically acceptable salt, stereoisomer, orprodrug thereof.

whereinR₁ is selected from the group consisting of CF₃, CF₂H, CF₂CH₃,CF₂CH₂CH₃, methyl, isopropyl, isobutyl, cyclopentyl, CH₂OCH₃, SCH₃,benzyl, thiophen-2-yl, and thiophen-3-yl;R₂ is selected from H, F, Cl, methoxy, and cyano; andR₃ is selected from H, F, Cl, and methoxy.

In a further aspect of the invention, R₁ is selected from CF₃, CF₂H, andCF₂CH₃; and R₂ is hydrogen.

In a further aspect of the invention, R₁ is selected from the groupconsisting of CF₃, methyl, isopropyl, and isobutyl; and R₂ and R₃ areboth hydrogen.

In yet another aspect of the invention, R₁ is selected from the groupconsisting of CF₃, methyl, isopropyl, isobutyl, CF₂H, CF₂CH₃, andCF₂CH₂CH₃; and R₂ and R₃ are both hydrogen.

In a further aspect of the invention, suitable compounds of Formula Iinclude, but are not limited to:

-   4-(2-(difluoromethyl)-7-hydroxy-4-oxo-4H-chromen-3-yl)benzoic acid;-   4-(7-hydroxy-2-(methoxymethyl)-4-oxo-4H-chromen-3-yl)benzoic acid;-   4-(7-hydroxy-2-isopropyl-4-oxo-4H-chromen-3-yl)benzoic acid;-   4-(2-cyclopentyl-7-hydroxy-4-oxo-4H-chromen-3-yl)benzoic acid;-   4-(7-hydroxy-2-methyl-4-oxo-4H-chromen-3-yl)benzoic acid;-   4-(2-benzyl-7-hydroxy-4-oxo-4H-chromen-3-yl)benzoic acid;-   4-(7-hydroxy-4-oxo-2-(thiophen-2-yl)-4H-chromen-3-yl)benzoic acid;-   4-(7-hydroxy-4-oxo-2-(thiophen-3-yl)-4H-chromen-3-yl)benzoic acid;-   4-(7-hydroxy-2-isobutyl-4-oxo-4H-chromen-3-yl)benzoic acid;-   4-(7-hydroxy-4-oxo-2-(trifluoromethyl)-4H-chromen-3-yl)benzoic acid;-   4-(7-hydroxy-2-(methylthio)-4-oxo-4H-chromen-3-yl)benzoic acid;-   4-(6-chloro-7-hydroxy-4-oxo-2-(trifluoromethyl)-4H-chromen-3-yl)benzoic    acid;-   4-(6-fluoro-7-hydroxy-4-oxo-2-(trifluoromethyl)-4H-chromen-3-yl)benzoic    acid;-   2-fluoro-4-(7-hydroxy-4-oxo-2-(trifluoromethyl)-4H-chromen-3-yl)benzoic    acid;-   3-chloro-4-(7-hydroxy-4-oxo-2-(trifluoromethyl)-4H-chromen-3-yl)benzoic    acid;-   4-(2-(1,1-difluoroethyl)-7-hydroxy-4-oxo-4H-chromen-3-yl)benzoic    acid;-   4-(7-hydroxy-6-methoxy-4-oxo-2-(trifluoromethyl)-4H-chromen-3-yl)benzoic    acid;-   2-chloro-4-(7-hydroxy-4-oxo-2-(trifluoromethyl)-4H-chromen-3-yl)benzoic    acid;-   4-(2-(1,1-difluoropropyl)-7-hydroxy-4-oxo-4H-chromen-3-yl)benzoic    acid;-   4-(7-hydroxy-4-oxo-2-(trifluoromethyl)-4H-chromen-3-yl)-3-methoxybenzoic    acid; and-   4-(6-cyano-7-hydroxy-4-oxo-2-(trifluoromethyl)-4H-chromen-3-yl)benzoic    acid.

Further, in any of the compositions described herein, one or morecompounds or subgenus of compounds can be specifically excluded. In oneembodiment of the invention,4-(7-hydroxy-4-oxo-2-(trifluoromethyl)-4H-chromen-3-yl)benzoic acid isspecifically excluded.

Substituted chromone analogs are potent inhibitors of GSNOR. As used inthis context, the term “analog” refers to a compound having similarchemical structure and function as compounds of Formula I that retainsthe chromone core.

When a bond to a substituent is shown to cross a bond connecting twoatoms in a ring, then such substituent may be bonded to any atom in thering. When a substituent is listed without indicating the atom via whichsuch substituent is bonded to the rest of the compound of a givenformula, then such substituent may be bonded via any atom in suchsubstituent. Combinations of substituents and/or variables arepermissible, but only if such combinations result in stable compounds.

Some chromone analogs of the invention can also exist in variousisomeric forms, including configurational, geometric and conformationalisomers, as well as existing in various tautomeric forms, particularlythose that differ in the point of attachment of a hydrogen atom. As usedherein, the term “isomer” is intended to encompass all isomeric forms ofa compound including tautomeric forms of the compound. All tautomers ofshown or described compounds are also considered to be part of thepresent invention. Cis and trans geometric isomers of the compounds ofthe present invention are described and may be isolated as a mixture ofisomers or as separated isomeric forms. Many geometric isomers ofolefins, C═N double bonds, and the like can also be present in thecompounds described herein, and all such stable isomers are contemplatedin the present invention. All chiral, diastereomeric, racemic, andgeometric isomeric forms of a structure are intended, unless thespecific stereochemistry or isomeric form is specifically indicated.

The compounds described herein may have asymmetric centers. Compounds ofthe present invention containing an asymmetrically substituted atom maybe isolated in optically active or racemic forms. It is well known inthe art how to prepare optically active forms, such as by resolution ofracemic forms or by synthesis from optically active starting materials.

It is to be understood that isomers arising from such asymmetry (e.g.,all enantiomers and diastereomers) are included within the scope of theinvention, unless indicated otherwise. Such isomers can be obtained insubstantially pure form by classical separation techniques and bystereochemically controlled synthesis. Furthermore, the structures andother compounds and moieties discussed in this application also includeall tautomers thereof. Alkenes can include either the E- or Z-geometry,where appropriate.

It should be noted that if there is a discrepancy between a depictedstructure and a name given to that structure, the depicted structurecontrols. In addition, if the stereochemistry of a structure or aportion of a structure is not indicated with, for example, bold, wedged,or dashed lines, the structure or portion of the structure is to beinterpreted as encompassing all stereoisomers of the described compound.

2. Representative GSNOR Inhibitors

Examples 1-21 list representative novel chromone analogs of Formula I.The synthetic methods that can be used to prepare each compound aredetailed in Examples 1-21, with reference to the synthetic schemedepicted before Example 1, and reference to intermediates described inExample 23. Supporting mass spectrometry data and proton NMR data foreach compound is also included in Examples 1-21. GSNOR inhibitoractivity was determined by the assay described in Example 24 and IC₅₀values were obtained. GSNOR inhibitor compounds Examples 1-21 had anIC₅₀ of about <5 μM. GSNOR inhibitor compounds Examples 1, 2, 3, 5, 6,10, 13, 14, 15, 16, 18, 19, 20 had an IC₅₀ of about <0.5 μM. GSNORinhibitor compounds Examples 1, 10, 14, 15, 16, 18, and 20 had an IC₅₀of about <0.1 μM.

C. Definitions

As used herein, “about” will be understood by persons of ordinary skillin the art and will vary to some extent on the context in which it isused. If there are uses of the term which are not clear to persons ofordinary skill in the art given the context in which it is used, “about”will mean up to plus or minus 10% of the particular term.

As used herein, the term “bioactivity” indicates an effect on one ormore cellular or extracellular process (e.g., via binding, signaling,etc.) which can impact physiological or pathophysiological processes.

As used herein and unless otherwise indicated, the term “stereoisomer”means one stereoisomer of a compound that is substantially free of otherstereoisomers of that compound. For example, a stereomerically purecompound having one chiral center will be substantially free of theopposite enantiomer of the compound. A stereomerically pure compoundhaving two chiral centers will be substantially free of otherdiastereomers of the compound. In some embodiments, a stereomericallypure compound comprises greater than about 80% by weight of onestereoisomer of the compound and less than about 20% by weight of otherstereoisomers of the compound, for example greater than about 90% byweight of one stereoisomer of the compound and less than about 10% byweight of the other stereoisomers of the compound, or greater than about95% by weight of one stereoisomer of the compound and less than about 5%by weight of the other stereoisomers of the compound, or greater thanabout 97% by weight of one stereoisomer of the compound and less thanabout 3% by weight of the other stereoisomers of the compound.

As used herein, “protein” is used synonymously with “peptide,”“polypeptide,” or “peptide fragment.” A “purified” polypeptide, protein,peptide, or peptide fragment is substantially free of cellular materialor other contaminating proteins from the cell, tissue, or cell-freesource from which the amino acid sequence is obtained, or substantiallyfree from chemical precursors or other chemicals when chemicallysynthesized.

As used herein, “modulate” is meant to refer to an increase or decreasethe levels of a peptide or polypeptide, or to increase or decrease thestability or activity of a peptide or a polypeptide. The term “inhibit”is meant to refer to a decrease in the levels of a peptide or apolypeptide or to decrease in the stability or activity of a peptide ora polypeptide. In preferred embodiments, the peptide which is modulatedor inhibited is S-nitrosoglutathione (GSNO) or protein S-nitrosothiols(SNOs).

As used here, the terms “nitric oxide” and “NO” encompass unchargednitric oxide and charged nitric oxide species, particularly includingnitrosonium ion (NO′) and nitroxyl ion (NO⁻). The reactive form ofnitric oxide can be provided by gaseous nitric oxide. Compounds havingthe structure X—NO_(y) wherein X is a nitric oxide releasing, deliveringor transferring moiety, including any and all such compounds whichprovide nitric oxide to its intended site of action in a form active fortheir intended purpose, and Y is 1 or 2.

As utilized herein, the term “pharmaceutically acceptable” meansapproved by a regulatory agency of a federal or a state government orlisted in the U.S. Pharmacopoeia or other generally recognizedpharmacopoeia for use in animals and, more particularly, in humans. Theterm “carrier” refers to a diluent, adjuvant, excipient, or vehicle withwhich the therapeutic is administered and includes, but is not limitedto such sterile liquids as water and oils.

A “pharmaceutically acceptable salt” or “salt” of a GSNOR inhibitor is aproduct of the disclosed compound that contains an ionic bond, and istypically produced by reacting the disclosed compound with either anacid or a base, suitable for administering to a subject. Apharmaceutically acceptable salt can include, but is not limited to,acid addition salts including hydrochlorides, hydrobromides, phosphates,sulphates, hydrogen sulphates, alkylsulphonates, arylsulphonates,arylalkylsulfonates, acetates, benzoates, citrates, maleates, fumarates,succinates, lactates, and tartrates; alkali metal cations such as Li,Na, K, alkali earth metal salts such as Mg or Ca, or organic aminesalts.

A “pharmaceutical composition” is a formulation comprising the disclosedcompounds in a form suitable for administration to a subject. Apharmaceutical composition of the invention is preferably formulated tobe compatible with its intended route of administration. Examples ofroutes of administration include, but are not limited to, oral andparenteral, e.g., intravenous, intradermal, subcutaneous, inhalation,topical, transdermal, transmucosal, and rectal administration.

“Stable compound” and “stable structure” are meant to indicate acompound that is sufficiently robust to survive isolation to a usefuldegree of purity from a reaction mixture, and formulation into anefficacious therapeutic agent.

As used herein the term “therapeutically effective amount” generallymeans the amount necessary to ameliorate at least one symptom of adisorder to be prevented, reduced, or treated as described herein. Thephrase “therapeutically effective amount” as it relates to the GSNORinhibitors of the present invention shall mean the GSNOR inhibitordosage that provides the specific pharmacological response for which theGSNOR inhibitor is administered in a significant number of subjects inneed of such treatment. It is emphasized that a therapeuticallyeffective amount of a GSNOR inhibitor that is administered to aparticular subject in a particular instance will not always be effectivein treating the conditions/diseases described herein, even though suchdosage is deemed to be a therapeutically effective amount by those ofskill in the art.

The term “biological sample” includes, but is not limited to, samples ofblood (e.g., serum, plasma, or whole blood), urine, saliva, sweat,breast milk, vaginal secretions, semen, hair follicles, skin, teeth,bones, nails, or other secretions, body fluids, tissues, or cells. Inaccordance with the invention, the levels of the S-nitrosoglutathionereductase in the biological sample can be determined by the methodsdescribed in U.S. Patent Application Publication No. 2005/0014697.

D. Pharmaceutical Compositions Comprising a GSNOR Inhibitor

The invention encompasses pharmaceutical compositions comprising atleast one GSNOR inhibitor described herein and at least onepharmaceutically acceptable carrier. Suitable carriers are described in“Remington: The Science and Practice, Twentieth Edition,” published byLippincott Williams & Wilkins, which is incorporated herein byreference. Pharmaceutical compositions according to the invention mayalso comprise one or more non-GSNOR inhibitor active agents.

The pharmaceutical compositions of the invention can comprise novelGSNOR inhibitors described herein, the pharmaceutical compositions cancomprise known compounds which previously were not know to have GSNORinhibitor activity, or a combination thereof.

The GSNOR inhibitors can be utilized in any pharmaceutically acceptabledosage form, including but not limited to injectable dosage forms,liquid dispersions, gels, aerosols, ointments, creams, lyophilizedformulations, dry powders, tablets, capsules, controlled releaseformulations, fast melt formulations, delayed release formulations,extended release formulations, pulsatile release formulations, mixedimmediate release and controlled release formulations, etc.Specifically, the GSNOR inhibitors described herein can be formulated:(a) for administration selected from the group consisting of oral,pulmonary, intravenous, intra-arterial, intrathecal, intra-articular,rectal, ophthalmic, colonic, parenteral, intracisternal, intravaginal,intraperitoneal, local, buccal, nasal, and topical administration; (b)into a dosage form selected from the group consisting of liquiddispersions, gels, aerosols, ointments, creams, tablets, sachets andcapsules; (c) into a dosage form selected from the group consisting oflyophilized formulations, dry powders, fast melt formulations,controlled release formulations, delayed release formulations, extendedrelease formulations, pulsatile release formulations, and mixedimmediate release and controlled release formulations; or (d) anycombination thereof.

For respiratory infections, an inhalation formulation can be used toachieve high local concentrations. Formulations suitable for inhalationinclude dry power or aerosolized or vaporized solutions, dispersions, orsuspensions capable of being dispensed by an inhaler or nebulizer intothe endobronchial or nasal cavity of infected patients to treat upperand lower respiratory bacterial infections.

Solutions or suspensions used for parenteral, intradermal, orsubcutaneous application can comprise one or more of the followingcomponents: (1) a sterile diluent such as water for injection, salinesolution, fixed oils, polyethylene glycols, glycerine, propylene glycolor other synthetic solvents; (2) antibacterial agents such as benzylalcohol or methyl parabens; (3) antioxidants such as ascorbic acid orsodium bisulfite; (4) chelating agents such asethylenediaminetetraacetic acid; (5) buffers such as acetates, citratesor phosphates; and (5) agents for the adjustment of tonicity such assodium chloride or dextrose. The pH can be adjusted with acids or bases,such as hydrochloric acid or sodium hydroxide. A parenteral preparationcan be enclosed in ampoules, disposable syringes or multiple dose vialsmade of glass or plastic.

Pharmaceutical compositions suitable for injectable use may comprisesterile aqueous solutions (where water soluble) or dispersions andsterile powders for the extemporaneous preparation of sterile injectablesolutions or dispersion. For intravenous administration, suitablecarriers include physiological saline, bacteriostatic water, CremophorEL (BASF, Parsippany, N.J.) or phosphate buffered saline (PBS). In allcases, the composition must be sterile and should be fluid to the extentthat easy syringability exists. The pharmaceutical composition should bestable under the conditions of manufacture and storage and should bepreserved against the contaminating action of microorganisms such asbacteria and fungi.

The carrier can be a solvent or dispersion medium comprising, forexample, water, ethanol, polyol (for example, glycerol, propyleneglycol, and liquid polyethylene glycol, and the like), and suitablemixtures thereof. The proper fluidity can be maintained, for example, bythe use of a coating such as lecithin, by the maintenance of therequired particle size in the case of dispersion and by the use ofsurfactants. Prevention of the action of microorganisms can be achievedby various antibacterial and antifungal agents, for example, parabens,chlorobutanol, phenol, ascorbic acid, thimerosal, and the like. In manycases, it will be preferable to include isotonic agents, for example,sugars, polyalcohols such as mannitol or sorbitol, and inorganic saltssuch as sodium chloride in the composition. Prolonged absorption of theinjectable compositions can be brought about by including in thecomposition an agent which delays absorption, for example, aluminummonostearate and gelatin.

Sterile injectable solutions can be prepared by incorporating the activereagent (e.g., GSNOR inhibitor) in the required amount in an appropriatesolvent with one or a combination of ingredients enumerated above, asrequired, followed by filtered sterilization. Generally, dispersions areprepared by incorporating at least one GSNOR inhibitor into a sterilevehicle that contains a basic dispersion medium and any other requiredingredients. In the case of sterile powders for the preparation ofsterile injectable solutions, exemplary methods of preparation includevacuum drying and freeze-drying, both of which yield a powder of theGSNOR inhibitor plus any additional desired ingredient from a previouslysterile-filtered solution thereof.

Oral compositions generally include an inert diluent or an ediblecarrier. They can be enclosed, for example, in gelatin capsules orcompressed into tablets. For the purpose of oral therapeuticadministration, the GSNOR inhibitor can be incorporated with excipientsand used in the form of tablets, troches, or capsules. Oral compositionscan also be prepared using a fluid carrier for use as a mouthwash,wherein the compound in the fluid carrier is applied orally and swishedand expectorated or swallowed. Pharmaceutically compatible bindingagents, and/or adjuvant materials can be included as part of thecomposition.

For administration by inhalation, the compounds are delivered in theform of an aerosol spray from pressured container or dispenser thatcontains a suitable propellant, e.g., a gas such as carbon dioxide, anebulized liquid, or a dry powder from a suitable device. Fortransmucosal or transdermal administration, penetrants appropriate tothe barrier to be permeated are used in the formulation. Such penetrantsare generally known in the art, and include, for example, fortransmucosal administration, detergents, bile salts, and fusidic acidderivatives. Transmucosal administration can be accomplished through theuse of nasal sprays or suppositories. For transdermal administration,the active reagents are formulated into ointments, salves, gels, orcreams as generally known in the art. The reagents can also be preparedin the form of suppositories (e.g., with conventional suppository basessuch as cocoa butter and other glycerides) or retention enemas forrectal delivery.

In one embodiment, the GSNOR inhibitors are prepared with carriers thatwill protect against rapid elimination from the body. For example, acontrolled release formulation can be used, including implants andmicroencapsulated delivery systems. Biodegradable, biocompatiblepolymers can be used, such as ethylene vinyl acetate, polyanhydrides,polyglycolic acid, collagen, polyorthoesters, and polylactic acid.Methods for preparation of such formulations will be apparent to thoseskilled in the art. The materials can also be obtained commercially.

Liposomal suspensions (including liposomes targeted to infected cellswith monoclonal antibodies to viral antigens) can also be used aspharmaceutically acceptable carriers. These can be prepared according tomethods known to those skilled in the art, for example, as described inU.S. Pat. No. 4,522,811.

Additionally, suspensions of the GSNOR inhibitors may be prepared asappropriate oily injection suspensions. Suitable lipophilic solvents orvehicles include fatty oils, such as sesame oil, or synthetic fatty acidesters, such as ethyl oleate, triglycerides, or liposomes. Non-lipidpolycationic amino polymers may also be used for delivery. Optionally,the suspension may also include suitable stabilizers or agents toincrease the solubility of the compounds and allow for the preparationof highly concentrated solutions.

It is especially advantageous to formulate oral or parenteralcompositions in dosage unit form for ease of administration anduniformity of dosage. Dosage unit form as used herein refers tophysically discrete units suited as unitary dosages for the subject tobe treated; each unit containing a predetermined quantity of GSNORinhibitor calculated to produce the desired therapeutic effect inassociation with the required pharmaceutical carrier. The specificationfor the dosage unit forms of the invention are dictated by and directlydependent on the unique characteristics of the GSNOR inhibitor and theparticular therapeutic effect to be achieved, and the limitationsinherent in the art of compounding such an active agent for thetreatment of individuals.

Pharmaceutical compositions according to the invention comprising atleast one GSNOR inhibitor can comprise one or more pharmaceuticalexcipients. Examples of such excipients include, but are not limited tobinding agents, filling agents, lubricating agents, suspending agents,sweeteners, flavoring agents, preservatives, buffers, wetting agents,disintegrants, effervescent agents, and other excipients. Suchexcipients are known in the art. Exemplary excipients include: (1)binding agents which include various celluloses and cross-linkedpolyvinylpyrrolidone, microcrystalline cellulose, such as Avicel® PH101and Avicel® PH102, silicified microcrystalline cellulose (ProSolvSMCC™), gum tragacanth and gelatin; (2) filling agents such as variousstarches, lactose, lactose monohydrate, and lactose anhydrous; (3)disintegrating agents such as alginic acid, Primogel, corn starch,lightly crosslinked polyvinyl pyrrolidone, potato starch, maize starch,and modified starches, croscarmellose sodium, cross-povidone, sodiumstarch glycolate, and mixtures thereof; (4) lubricants, including agentsthat act on the flowability of a powder to be compressed, includemagnesium stearate, colloidal silicon dioxide, such as Aerosil® 200,talc, stearic acid, calcium stearate, and silica gel; (5) glidants suchas colloidal silicon dioxide; (6) preservatives, such as potassiumsorbate, methylparaben, propylparaben, benzoic acid and its salts, otheresters of parahydroxybenzoic acid such as butylparaben, alcohols such asethyl or benzyl alcohol, phenolic compounds such as phenol, orquaternary compounds such as benzalkonium chloride; (7) diluents such aspharmaceutically acceptable inert fillers, such as microcrystallinecellulose, lactose, dibasic calcium phosphate, saccharides, and/ormixtures of any of the foregoing; examples of diluents includemicrocrystalline cellulose, such as Avicel® PH101 and Avicel® PH102;lactose such as lactose monohydrate, lactose anhydrous, and Pharmatose®DCL21; dibasic calcium phosphate such as Emcompress®; mannitol; starch;sorbitol; sucrose; and glucose; (8) sweetening agents, including anynatural or artificial sweetener, such as sucrose, saccharin sucrose,xylitol, sodium saccharin, cyclamate, aspartame, and acesulfame; (9)flavoring agents, such as peppermint, methyl salicylate, orangeflavoring, Magnasweet® (trademark of MAFCO), bubble gum flavor, fruitflavors, and the like; and (10) effervescent agents, includingeffervescent couples such as an organic acid and a carbonate orbicarbonate. Suitable organic acids include, for example, citric,tartaric, malic, fumaric, adipic, succinic, and alginic acids andanhydrides and acid salts. Suitable carbonates and bicarbonates include,for example, sodium carbonate, sodium bicarbonate, potassium carbonate,potassium bicarbonate, magnesium carbonate, sodium glycine carbonate,L-lysine carbonate, and arginine carbonate. Alternatively, only thesodium bicarbonate component of the effervescent couple may be present.

E. Kits Comprising the Compositions of the Invention

The present invention also encompasses kits comprising the compositionsof the invention. Such kits can comprise, for example, (1) at least oneGSNOR inhibitor; and (2) at least one pharmaceutically acceptablecarrier, such as a solvent or solution. Additional kit components canoptionally include, for example: (1) any of the pharmaceuticallyacceptable excipients identified herein, such as stabilizers, buffers,etc., (2) at least one container, vial or similar apparatus for holdingand/or mixing the kit components; and (3) delivery apparatus, such as aninhaler, nebulizer, syringe, etc.

F. Methods of Preparing GSNOR Inhibitors

The GSNOR inhibitors of the invention can readily be synthesized usingknown synthetic methodologies or via a modification of known syntheticmethodologies. As would be readily recognized by a skilled artisan, themethodologies described below allow the synthesis of chromones having avariety of substituents. Exemplary synthetic methods are described inthe examples below.

If needed, further purification and separation of enantiomers anddiastereomers can be achieved by routine procedures known in the art.Thus, for example, the separation of enantiomers of a compound can beachieved by the use of chiral HPLC and related chromatographictechniques. Diastereomers can be similarly separated. In some instances,however, diastereomers can simply be separated physically, such as, forexample, by controlled precipitation or crystallization.

The process of the invention, when carried out as prescribed herein, canbe conveniently performed at temperatures that are routinely accessiblein the art. In one embodiment, the process is performed at a temperaturein the range of about 25° C. to about 110° C. In another embodiment, thetemperature is in the range of about 40° C. to about 100° C. In yetanother embodiment, the temperature is in the range of about 50° C. toabout 95° C.

Synthetic steps that require a base are carried out using any convenientorganic or inorganic base. Typically, the base is not nucleophilic.Thus, in one embodiment, the base is selected from carbonates,phosphates, hydroxides, alkoxides, salts of disilazanes, and tertiaryamines.

The process of the invention, when performed as described herein, can besubstantially complete after several minutes to after several hoursdepending upon the nature and quantity of reactants and reactiontemperature. The determination of when the reaction is substantiallycomplete can be conveniently evaluated by ordinary techniques known inthe art such as, for example, HPLC, LCMS, TLC, and ¹H NMR.

G. Method of Treatment

The invention encompasses methods of preventing or treating (e.g.,alleviating one or more symptoms of) medical conditions through use ofone or more of the disclosed compounds. The methods compriseadministering a therapeutically effective amount of a GSNOR inhibitor toa patient in need. The compositions of the invention can also be usedfor prophylactic therapy.

The GSNOR inhibitor used in the methods of treatment according to theinvention can be: (1) a novel GSNOR inhibitor described herein, or apharmaceutically acceptable salt thereof, a prodrug thereof, or ametabolite thereof; (2) a compound which was known prior to the presentinvention, but wherein it was not known that the compound is a GSNORinhibitor, or a pharmaceutically acceptable salt thereof, a prodrugthereof, or a metabolite thereof; or (3) a compound which was knownprior to the present invention, and wherein it was known that thecompound is a GSNOR inhibitor, but wherein it was not known that thecompound is useful for the methods of treatment described herein, or apharmaceutically acceptable salt thereof, a prodrug thereof, or ametabolite thereof.

The patient can be any animal, domestic, livestock or wild, including,but not limited to cats, dogs, horses, pigs and cattle, and preferablyhuman patients. As used herein, the terms patient and subject may beused interchangeably.

In subjects with deleteriously high levels of GSNOR or GSNOR activity,modulation may be achieved, for example, by administering one or more ofthe disclosed compounds that disrupts or down-regulates GSNOR function,or decreases GSNOR levels. These compounds may be administered withother GSNOR inhibitor agents, such as anti-GSNOR antibodies or antibodyfragments, GSNOR antisense, siRNA, or small molecules, or otherinhibitors, alone or in combination with other agents as described indetail herein.

The present invention provides a method of treating a subject afflictedwith a disorder ameliorated by NO donor therapy. Such a method comprisesadministering to a subject a therapeutically effective amount of a GSNORinhibitor.

As used herein, “treating” describes the management and care of apatient for the purpose of combating a disease, condition, or disorderand includes the administration of a compound of the present inventionto prevent the onset of the symptoms or complications, alleviating thesymptoms or complications, or eliminating the disease, condition ordisorder. More specifically, “treating” includes reversing, attenuating,alleviating, minimizing, suppressing or halting at least one deleterioussymptom or effect of a disease (disorder) state, disease progression,disease causative agent (e.g., bacteria or viruses), or other abnormalcondition. Treatment is continued as long as symptoms and/or pathologyameliorate.

The disorders can include pulmonary disorders associated with hypoxemiaand/or smooth muscle constriction in the lungs and/or lung infectionand/or lung injury (e.g., pulmonary hypertension, ARDS, asthma,pneumonia, pulmonary fibrosis/interstitial lung diseases, cysticfibrosis, COPD) cardiovascular disease and heart disease, includingconditions such as hypertension, ischemic coronary syndromes,atherosclerosis, heart failure, glaucoma, diseases characterized byangiogenesis (e.g., coronary artery disease), disorders where there isrisk of thrombosis occurring, disorders where there is risk ofrestenosis occurring, chronic inflammatory diseases (e.g., AID dementiaand psoriasis), diseases where there is risk of apoptosis occurring(e.g., heart failure, atherosclerosis, degenerative neurologicdisorders, arthritis and liver injury (ischemic or alcoholic)),impotence, obesity caused by eating in response to craving for food,stroke, reperfusion injury (e.g., traumatic muscle injury in heart orlung or crush injury), and disorders where preconditioning of heart orbrain for NO protection against subsequent ischemic events isbeneficial.

In one embodiment, the compounds of the present invention or apharmaceutically acceptable salt thereof, or a prodrug or metabolitethereof, can be administered in combination with an NO donor. An NOdonor donates nitric oxide or a related redox species and more generallyprovides nitric oxide bioactivity, that is activity which is identifiedwith nitric oxide, e.g., vasorelaxation or stimulation or inhibition ofa receptor protein, e.g., ras protein, adrenergic receptor, NFκB. NOdonors including S-nitroso, O-nitroso, C-nitroso and N-nitroso compoundsand nitro derivatives thereof and metal NO complexes, but not excludingother NO bioactivity generating compounds, useful herein are describedin “Methods in Nitric Oxide Research,” Feelisch et al. eds., pages71-115 (J. S., John Wiley & Sons, New York, 1996), which is incorporatedherein by reference. NO donors which are C-nitroso compounds wherenitroso is attached to a tertiary carbon which are useful herein includethose described in U.S. Pat. No. 6,359,182 and in WO 02/34705. Examplesof S-nitroso compounds, including S-nitrosothiols useful herein,include, for example, S-nitrosoglutathione,S-nitroso-N-acetylpenicillamine, S-nitroso-cysteine and ethyl esterthereof, S-nitroso cysteinyl glycine,S-nitroso-gamma-methyl-L-homocysteine, S-nitroso-L-homocysteine,S-nitroso-gamma-thio-L-leucine, S-nitroso-delta-thio-L-leucine, andS-nitrosoalbumin Examples of other NO donors useful herein are sodiumnitroprusside (nipride), ethyl nitrite, isosorbide, nitroglycerin, SIN 1which is molsidomine, furoxamines, N-hydroxy (N-nitrosamine) andperfluorocarbons that have been saturated with NO or a hydrophobic NOdonor.

The combination of a GSNOR inhibitor with R(+) enantiomer of amlodipine,a known NO releaser (Zhang X. P at al. 2002 J. CardiovascularPharmacology 39, 208-214) is also an embodiment of the presentinvention.

The present invention also provides a method of treating a subjectafflicted with pathologically proliferating cells where the methodcomprises administering to said subject a therapeutically effectiveamount of an inhibitor of GSNOR. The inhibitors of GSNOR are thecompounds as defined above, or a pharmaceutically acceptable saltthereof, or a prodrug or metabolite thereof, in combination with apharmaceutically acceptable carrier. Treatment is continued as long assymptoms and/or pathology ameliorate.

In another embodiment, the pathologically proliferating cells can bepathologically proliferating microbes. The microbes involved can bethose where GSNOR is expressed to protect the microbe from nitrosativestress or where a host cell infected with the microbe expresses theenzyme, thereby protecting the microbe from nitrosative stress. The term“pathologically proliferating microbes” is used herein to meanpathologic microorganisms including but not limited to pathologicbacteria, pathologic viruses, pathologic Chlamydia, pathologic protozoa,pathologic Rickettsia, pathologic fungi, and pathologic mycoplasmata.More detail on the applicable microbes is set forth at columns 11 and 12of U.S. Pat. No. 6,057,367. The term “host cells infected withpathologic microbes” includes not only mammalian cells infected withpathologic viruses but also mammalian cells containing intracellularbacteria or protozoa, e.g., macrophages containing Mycobacteriumtuberculosis, Mycobacterium leper (leprosy), or Salmonella typhi(typhoid fever).

In another embodiment, the pathologically proliferating cells can bepathologic helminths. The term “pathologic helminths” is used herein torefer to pathologic nematodes, pathologic trematodes and pathologiccestodes. More detail on the applicable helminths is set forth at column12 of U.S. Pat. No. 6,057,367.

In another embodiment, the pathologically proliferating cells can bepathologically proliferating mammalian cells. The term “pathologicallyproliferating mammalian cells” as used herein means cells of the mammalthat grow in size or number in said mammal so as to cause a deleteriouseffect in the mammal or its organs. The term includes, for example, thepathologically proliferating or enlarging cells causing restenosis, thepathologically proliferating or enlarging cells causing benign prostatichypertrophy, the pathologically proliferating cells causing myocardialhypertrophy and proliferating cells at inflammatory sites such assynovial cells in arthritis or cells associated with a cellproliferation disorder.

As used herein, the term “cell proliferative disorder” refers toconditions in which the unregulated and/or abnormal growth of cells canlead to the development of an unwanted condition or disease, which canbe cancerous or non-cancerous, for example a psoriatic condition. Asused herein, the term “psoriatic condition” refers to disordersinvolving keratinocyte hyperproliferation, inflammatory cellinfiltration, and cytokine alteration. The cell proliferative disordercan be a precancerous condition or cancer. The cancer can be primarycancer or metastatic cancer, or both.

As used herein, the term “cancer” includes solid tumors, such as lung,breast, colon, ovarian, pancreas, prostate, adenocarcinoma, squamouscarcinoma, sarcoma, malignant glioma, leiomyosarcoma, hepatoma, head andneck cancer, malignant melanoma, non-melanoma skin cancers, as well ashematologic tumors and/or malignancies, such as leukemia, childhoodleukemia and lymphomas, multiple myeloma, Hodgkin's disease, lymphomasof lymphocytic and cutaneous origin, acute and chronic leukemia such asacute lymphoblastic, acute myelocytic or chronic myelocytic leukemia,plasma cell neoplasm, lymphoid neoplasm and cancers associated withAIDS.

In addition to psoriatic conditions, the types of proliferative diseaseswhich may be treated using the compositions of the present invention areepidermic and dermoid cysts, lipomas, adenomas, capillary and cutaneoushemangiomas, lymphangiomas, nevi lesions, teratomas, nephromas,myofibromatosis, osteoplastic tumors, and other dysplastic masses andthe like. In one embodiment, proliferative diseases include dysplasiasand disorders of the like.

In one embodiment, the treating cancer comprises a reduction in tumorsize, decrease in tumor number, a delay of tumor growth, decrease inmetastaic lesions in other tissues or organs distant from the primarytumor site, an improvement in the survival of patients, or animprovement in the quality of patient life, or at least two of theabove.

In another embodiment, the treating a cell proliferative disordercomprises a reduction in the rate of cellular proliferation, reductionin the proportion of proliferating cells, a decrease in size of an areaor zone of cellular proliferation, or a decrease in the number orproportion of cells having an abnormal appearance or morphology, or atleast two of the above.

In yet another embodiment, the compounds of the present invention or apharmaceutically acceptable salt thereof, a prodrug thereof, ormetabolite thereof, can be administered in combination with a secondchemotherapeutic agent. In a further embodiment, the secondchemotherapeutic agent is selected from the group consisting oftamoxifen, raloxifene, anastrozole, exemestane, letrozole, cisplatin,carboplatin, paclitaxel, cyclophosphamide, lovastatin, minosine,gemcitabine, araC, 5-fluorouracil, methotrexate, docetaxel, goserelin,vincristine, vinblastin, nocodazole, teniposide, etoposide, epothilone,navelbine, camptothecin, daunonibicin, dactinomycin, mitoxantrone,amsacrine, doxorubicin, epirubicin, idarubicin imatanib, gefitinib,erlotinib, sorafenib, sunitinib malate, trastuzumab, rituximab,cetuximab, and bevacizumab.

In one embodiment, the compounds of the present invention or apharmaceutically acceptable salt thereof, a prodrug thereof, ormetabolite thereof, can be administered in combination with an agentthat imposes nitrosative or oxidative stress. Agents for selectivelyimposing nitrosative stress to inhibit proliferation of pathologicallyproliferating cells in combination therapy with GSNOR inhibitors hereinand dosages and routes of administration therefor include thosedisclosed in U.S. Pat. No. 6,057,367, which is incorporated herein.Supplemental agents for imposing oxidative stress (i.e., agents thatincrease GSSG (oxidized glutathione) over GSH (glutathione) ratio orNAD(P) over NAD(P)H ratio or increase thiobarbituric acid derivatives)in combination therapy with GS-FDH inhibitors herein include, forexample, L-buthionine-S-sulfoximine (BSO), glutathione reductaseinhibitors (e.g., BCNU), inhibitors or uncouplers of mitochondrialrespiration and drugs that increase reactive oxygen species (ROS), e.g.,adriamycin, in standard dosages with standard routes of administration.

GSNOR inhibitors may also be co-administered with a phosphodiesteraseinhibitor (e.g., rolipram, cilomilast, roflumilast, Viagra® (sildenifilcitrate), Cialis® (tadalafil), Levitra® (vardenifil), etc.) a β-agonist,a steroid, or a leukotriene antagonist (LTD-4). Those skilled in the artcan readily determine the appropriate therapeutically effective amountdepending on the disorder to be ameliorated.

GSNOR inhibitors may be used as a means to improve β-adrenergicsignaling. In particular, inhibitors of GSNOR alone or in combinationwith β-agonists could be used to treat or protect against heart failure,or other vascular disorders such as hypertension and asthma. GSNORinhibitors can also be used to modulate G protein coupled receptors(GPCRs) by potentiating Gs G-protein, leading to smooth musclerelaxation (e.g., airway and blood vessels), and by attenuating GqG-protein, and thereby preventing smooth muscle contraction (e.g., inairway and blood vessels).

The therapeutically effective amount for the treatment of a subjectafflicted with a disorder ameliorated by NO donor therapy is the GSNORinhibiting amount in vivo that causes amelioration of the disorder beingtreated or protects against a risk associated with the disorder. Forexample, for asthma, a therapeutically effective amount is abronchodilating effective amount; for cystic fibrosis, a therapeuticallyeffective amount is an airway obstruction ameliorating effective amount;for ARDS, a therapeutically effective amount is a hypoxemia amelioratingeffective amount; for heart disease, a therapeutically effective amountis an angina relieving or angiogenesis inducing effective amount; forhypertension, a therapeutically effective amount is a blood pressurereducing effective amount; for ischemic coronary disorders, atherapeutic amount is a blood flow increasing effective amount; foratherosclerosis, a therapeutically effective amount is an endothelialdysfunction reversing effective amount; for glaucoma, a therapeuticamount is an intraocular pressure reducing effective amount; fordiseases characterized by angiogenesis, a therapeutically effectiveamount is an angiogenesis inhibiting effective amount; for disorderswhere there is risk of thrombosis occurring, a therapeutically effectiveamount is a thrombosis preventing effective amount; for disorders wherethere is risk of restenosis occurring, a therapeutically effectiveamount is a restenosis inhibiting effective amount; for chronicinflammatory diseases, a therapeutically effective amount is aninflammation reducing effective amount; for disorders where there isrisk of apoptosis occurring, a therapeutically effective amount is anapoptosis preventing effective amount; for impotence, a therapeuticallyeffective is an erection attaining or sustaining effective amount; forobesity, a therapeutically effective amount is a satiety causingeffective amount; for stroke, a therapeutically effective amount is ablood flow increasing or a TIA protecting effective amount; forreperfusion injury, a therapeutically effective amount is a functionincreasing effective amount; and for preconditioning of heart and brain,a therapeutically effective amount is a cell protective effectiveamount, e.g., as measured by triponin or CPK.

The therapeutically effective amount for the treatment of a subjectafflicted with pathologically proliferating cells means a GSNORinhibiting amount in vivo which is an antiproliferative effectiveamount. Such antiproliferative effective amount as used herein means anamount causing reduction in rate of proliferation of at least about 20%,at least about 10%, at least about 5%, or at least about 1%.

In general, the dosage, i.e., the therapeutically effective amount,ranges from 1 μg to 10 g/kg and often ranges from 10 μg to 1 g/kg or 10μg to 100 mg/kg body weight of the subject being treated, per day.

H. Other Uses

The compounds of the present invention or a pharmaceutically acceptablesalt thereof, or a prodrug or metabolite thereof, can be applied tovarious apparatus in circumstances when the presence of such compoundswould be beneficial. Such apparatus can be any device or container, forexample, implantable devices in which a GSNOR inhibitor can be used tocoat a surgical mesh or cardiovascular stent prior to implantation in apatient. The GSNOR inhibitors of the present invention can also beapplied to various apparatus for in vitro assay purposes or forculturing cells.

The compounds of the present invention or a pharmaceutically acceptablesalt thereof, or a prodrug or metabolite thereof, can also be used as anagent for the development, isolation or purification of binding partnersto GSNOR inhibitor compounds, such as antibodies, natural ligands, andthe like. Those skilled in the art can readily determine related usesfor the compounds of the present invention.

EXAMPLES

The following Scheme and Examples are given to illustrate the presentinvention. It should be understood, however, that the invention is notto be limited to the specific conditions or details described in theseexamples. Throughout the specification, any and all references to apublicly available document, including a U.S. patent, are specificallyincorporated by reference.

The GSNOR inhibitors of the invention can readily be synthesized usingknown synthetic methodologies or via a modification of known syntheticmethodologies. As would be readily recognized by a skilled artisan, themethodologies described below allow the synthesis of chromones having avariety of substituents. General scheme I below is a representativeprocedure for making chromone compounds of the invention. Additionalsynthetic detail can be found in Examples 1-21, and in the Intermediatesection, Example 23.

Example 1 4-(2-(difluoromethyl)-7-hydroxy-4-oxo-4H-chromen-3-yl)benzoicacid

Synthesis:

Prepared following General Scheme 1, starting from Intermediate A-1a,using conditions a) (modified) and c).

Step 1: Synthesis of ethyl4-(2-(difluoromethyl)-7-hydroxy-4-oxo-4H-chromen-3-yl)benzoate

To a solution of Intermediate A-1a (450 mg, 1.5 mmol) and TEA (758 mg,7.5 mmol) was added 2,2-difluoroacetic anhydride (522 mg, 3.0 mmol). Themixture was stirred at 120° C. for 4 h. The reaction mixture was cooledto room temperature and concentrated in vacuo to afford a brown solid,which was used for next step without further purification (400 mg, 74%).

Step 2: Synthesis of4-(2-(difluoromethyl)-7-hydroxy-4-oxo-4H-chromen-3-yl)benzoic acid(Example 1), an Example of c) Conditions

To a mixture of ethyl4-(2-(difluoromethyl)-7-hydroxy-4-oxo-4H-chromen-3-yl)benzoate (400 mg,1.16 mmol) in 1,4-dioxane (1 mL) was added Conc. HCl (1 mL). Thereaction mixture was heated to reflux overnight. The mixture was cooledto room temperature and filtered. The filtered mass was washed withwater (5 mL) 2 times and ethanol (2 mL) and purified by prep-HPLC toafford a white solid (50 mg, 13%).

Data:

¹H NMR (500 MHz, DMSO-d₆, TMS): δ 13.11 (s, 1H), 11.09 (s, 1H), 8.02 (d,J=8.5 Hz, 2H), 7.95 (d, J=8.5 Hz, 1H), 7.43 (d, J=8.5 Hz, 2H), 7.01 (dd,J=2.5, 9.0 Hz, 1H), 6.96 (d, J=2.5 Hz, 1H), 6.68 (t, J=51.0 Hz, 1H); MS(ESI): m/z 333.0 [M+1]⁺.

Example 2 4-(7-hydroxy-2-(methoxymethyl)-4-oxo-4H-chromen-3-yl)benzoicacid

Synthesis:

Prepared following General Scheme 1, starting from Intermediate A-1a,using conditions b) (modified) and c).

Step 1: Synthesis of ethyl4-(7-hydroxy-2-(methoxymethyl)-4-oxo-4H-chromen-3-yl)benzoate, anExample of b) Conditions

To stirred mixture of Intermediate A-1a (450 mg, 1.5 mmol) and Et₃N (825mg, 7.5 mmol) in DCM (5 mL) was added 2-methoxyacetyl chloride (486 mg,4.5 mmol). Then the mixture was stirred at room temperature for 5 h. Thesolution was removed under reduced pressure and the residue was dilutedwith water (50 mL), extracted with ethyl acetate (50 mL×3). The organiclayers were dried over Na₂SO₄, concentrated and purified by silica gelcolumn chromatography (PE:EA=1:1) to afford ethyl4-(7-hydroxy-2-(methoxymethyl)-4-oxo-4H-chromen-3-yl)benzoate as ayellow oil (84 mg, 16%). MS (ESI): m/z 355.0 [M+1]⁺.

Step 2: Synthesis of4-(7-hydroxy-2-(methoxymethyl)-4-oxo-4H-chromen-3-yl)benzoic acid(Example 2)

General Scheme 1, c) conditions (purified by prep HPLC), see step 2 ofExample 1 for detailed example.

Data:

¹H NMR (MeOH-d₄, 500 MHz, TMS): δ 8.08 (d, J=7.5 Hz, 2H), 7.99 (d, J=9.0Hz, 1H), 7.41 (d, J=7.5 Hz, 2H), 6.94 (t, J=10.0 Hz, 1H), 6.89 (d, J=1.5Hz, 1H), 4.24 (s, 2H), 3.34 (s, 2H); MS (ESI): m/z 327.1 [M+1]⁺.

Example 3 4-(7-hydroxy-2-isopropyl-4-oxo-4H-chromen-3-yl)benzoic acid

Synthesis:

Prepared following General Scheme 1, starting from Intermediate A-1a,using conditions b) and d). Synthetic details included here as anexample of b) and d).

Step 1: Synthesis of ethyl4-(7-(isobutyryloxy)-2-isopropyl-4-oxo-4H-chromen-3-yl)benzoate, anExample of Scheme 1, b) Conditions

To a solution of Intermediate A-1a (450 mg, crude, 1.5 mmol) and TEA(834 μL, 6 mmol) in dried DCM (5 mL) was added dropwise isobutyrylchloride (480 μL, 4.5 mmol) at room temperature. The mixture was stirredfor 3 h. The volatiles were removed under reduced pressure. To theresidue was added TEA (5 mL) and heated at 95° C. overnight. Cooled toroom temperature and filtered; the filter cake was washed with EA (10mL). The filtrate was concentrated and purified by Combi-Flash (40 gsilica gel, start PE:EA=10:0 to 3:1 gradient, 40 mL/min, 40 min, 1.6 Ltotal solvent volume) to afford ethyl4-(7-(isobutyryloxy)-2-isopropyl-4-oxo-4H-chromen-3-yl)benzoate as awhite solid (80 mg, 13%).

Step 2: Synthesis of4-(7-hydroxy-2-isopropyl-4-oxo-4H-chromen-3-yl)benzoic acid (Example 3),an Example of Scheme 1, d) Conditions

To a solution of ethyl4-(7-(isobutyryloxy)-2-isopropyl-4-oxo-4H-chromen-3-yl)benzoate (80 mg,0.142 mmol) in THF (2 mL) was added a solution of lithium hydroxide (60mg, 1.42 mmol) in water (1 mL). The mixture was stirred at roomtemperature for 3 h. TLC (PE:EA=3:1) indicated that the reaction iscomplete. The organic solvents were removed under reduced pressure, thebasic water layers was extracted with DCM (10 mL×2) and adjusted to pH4˜5 with 1 N HCl solution, the precipitate was collected by filtration,dried in vacuo to give Example 3 as a yellow solid (58 mg, 94%).

Data:

¹H NMR (DMSO-d₆, 500 MHz, TMS): δ 12.99 (s, 1H), 10.80 (s, 1H), 7.99 (d,J=8 Hz, 2H), 7.87 (d, J=8.5 Hz, 1H), 7.38 (d, J=7.5 Hz, 2H), 6.92 (dd,J=8.5 Hz, 2 Hz, 1H), 6.88 (d, J=2 Hz, 1H), 2.75 (m, 1H), 1.21 (s, 3H),1.19 (s, 3H); MS (ESI): m/z 325.1 [M+1]⁺.

Example 4 4-(2-cyclopentyl-7-hydroxy-4-oxo-4H-chromen-3-yl)benzoic acid

Synthesis:

Prepared following General Scheme 1, starting from Intermediate A-1a andcyclopentanecarbonyl chloride in step 1 using condition b) to give ethyl4-(7-(cyclopentanecarbonyloxy)-2-cyclopentyl-4-oxo-4H-chromen-3-yl)benzoate.Step 2 followed d) conditions. See Example 3 for detailed example.

Data:

¹H NMR (DMSO-d₆, 500 MHz, TMS): δ 12.98 (brs, 1H), 10.79 (brs, 1H), 7.99(d, J=8.5 Hz, 2H), 7.87 (d, J=9 Hz, 1H), 7.37 (d, J=8 Hz, 2H), 6.91 (dd,J=9 Hz, 2 Hz, 1H), 6.85 (d, J=2 Hz, 1H), 2.84 (m, 1H), 1.78-1.84 (m,6H), 1.543 (s, 2H); MS (ESI): m/z 351.1 [M+1]⁺.

Example 5 4-(7-hydroxy-2-methyl-4-oxo-4H-chromen-3-yl)benzoic acid

Synthesis:

Prepared following General Scheme 1, starting from Intermediate A-1a andacetic anhydride in step 1 using condition a) (where reaction wasrefluxed for 3 hours after stirring for 2 h at room temperature, and waspurified by prep-TLC (PE:EA=3:1)). Step 2 followed c) conditions. SeeExample 1 for detailed example.

Data:

¹H NMR (MeOH-d₄, 500 MHz, TMS): δ 8.12 (d, J=8.0 Hz, 2H), 7.99 (d, J=8.5Hz, 1H), 7.43 (d, J=8.0 Hz, 2H), 6.94 (d, J=9.0 Hz, 1H), 6.88 (s, 1H),2.32 (s, 3H); MS (ESI): m/z 297.1 [M+1]⁺.

Example 6 4-(2-benzyl-7-hydroxy-4-oxo-4H-chromen-3-yl)benzoic acid

Synthesis:

Prepared following General Scheme 1, starting from Intermediate A-1a and2-phenylacetyl chloride in Step 1 using condition b) (where reaction wasrun at room temperature for 3 hours) to give ethyl4-(2-benzyl-7-hydroxy-4-oxo-4H-chromen-3-yl)benzoate. Step 2: followedd) conditions (where reaction was allowed to stir at room temperatureovernight, and was purified by prep-HPLC. See Example 3 for detailedexample.

Data:

¹H NMR (MeOH-d₄, 500 MHz): δ 8.12 (d, J=8.0 Hz, 2H), 7.99 (d, J=9.0 Hz,1H), 7.40 (d, J=8.0 Hz, 2H), 7.29 (q, J=14.5 Hz, 2H), 7.24 (d, J=7.5 Hz,2H), 7.22 (s, 1H), 7.15 (d, J=6.0 Hz, 2H), 6.94 (q, J=8.5 Hz, 1H), 6.83(s, 1H), 3.92 (s, 2H); MS (ESI): m/z 373.1 [M+1]⁺.

Example 7 4-(7-hydroxy-4-oxo-2-(thiophen-2-yl)-4H-chromen-3-yl)benzoicacid

Synthesis:

Prepared following General Scheme 1, starting from Intermediate A-1a andthiophene-2-carbonyl chloride in step 1 using condition b) to give3-(4-(ethoxycarbonyl)phenyl)-4-oxo-2-(thiophen-2-yl)-4H-chromen-7-ylthiophene-2-carboxylate. Step 2: followed d) conditions. See Example 3for detailed example.

Data:

¹H NMR (DMSO-d₆, 500 MHz, TMS): δ 13.08 (brs, 1H), 10.89 (s, 1H), 8.03(d, J=8 Hz, 2H), 7.90 (d, J=9 Hz, 1H), 7.77 (dd, J=5, 1 Hz, 1H), 7.41(d, J=8.5 Hz, 2H), 7.31 (dd, J=3.5, 1 Hz, 1H), 7.08 (m, 1H), 6.94˜6.98(m, 2H); MS (ESI): m/z 365.0 [M+1]⁺.

Example 8 4-(7-hydroxy-4-oxo-2-(thiophen-3-yl)-4H-chromen-3-yl)benzoicacid

Synthesis:

Prepared following General Scheme 1, starting from Intermediate A-1a andthiophene-3-carbonyl chloride in step 1 using condition b) to3-(4-(ethoxycarbonyl)phenyl)-4-oxo-2-(thiophen-3-yl)-4H-chromen-7-ylthiophene-3-carboxylate.Step 2 followed d) conditions. See Example 3 for detailed example.

Data:

¹H NMR (DMSO-d₆, 500 MHz, TMS): δ 13.01 (brs, 1H), 10.87 (s, 1H), 7.97(d, J=8 Hz, 2H), 7.92 (d, J=8.5 Hz, 1H), 7.78˜7.79 (m, 1H), 7.49˜7.51(m, 1H), 7.35 (d, J=8.5 Hz, 2H), 6.98 (d, J=2 Hz, 1H), 6.95 (dd, J=9, 2Hz, 1H), 6.70 (dd, J=5.5, 1.5 Hz, 1H); MS (ESI): m/z 365.0 [M+1]⁺.

Example 9 4-(7-hydroxy-2-isobutyl-4-oxo-4H-chromen-3-yl)benzoic acid

Synthesis:

Prepared following General Scheme 1, starting from Intermediate A-1a and3-methylbutanoyl chloride in Step 1 using condition b) to give ethyl4-(2-isobutyl-7-(3-methylbutanoyloxy)-4-oxo-4H-chromen-3-yl)benzoate.Step 2 followed d) conditions, with purification by recrystallizationfrom PE. See Example 3 for detailed example.

Data:

¹H NMR (MeOH-d₄, 500 MHz, TMS): δ 8.12 (d, J=8.0 Hz, 2H), 8.00 (d, J=9.0Hz, 1H), 7.39 (d, J=8.0 Hz, 2H), 6.95 (dd, J=2.5, 9.0 Hz, 1H), 6.88 (d,J=2.0 Hz, 1H), 2.49 (d, J=7.0 Hz, 2H), 2.40-2.20 (m, 1H), 0.90 (d, J=7.0Hz, 6H); MS (ESI): m/z 339.1 [M+1]⁺.

Example 104-(7-hydroxy-4-oxo-2-(trifluoromethyl)-4H-chromen-3-yl)benzoic acid

Synthesis:

Prepared following General Scheme 1, starting from Intermediate A-1a,using conditions a) and c). Synthetic details included here as anotherexample of a) and c).

Step 1: Synthesis of ethyl4-(7-hydroxy-4-oxo-2-(trifluoromethyl)-4H-chromen-3-yl)benzoate, Exampleof a) Conditions

To a solution of Intermediate A-1a (400 mg, 1.33 mmol) in DCM (10 mL)was added TFAA (1.39 g, 6.65 mmol) at 5˜10° C. After addition themixture was stirred at room temperature for 1 h, concentrated andpurified by prep-TLC (PE:EA=2:1) to afford product as a yellow solid(280 mg, 56%).

Step 2: Synthesis of4-(7-hydroxy-4-oxo-2-(trifluoromethyl)-4H-chromen-3-yl)benzoic acid(Example 10), an Example of c) Conditions

To a solution of ethyl4-(7-hydroxy-4-oxo-2-(trifluoromethyl)-4H-chromen-3-yl)benzoate (23 mg,0.06 mmol) in dioxane (0.5 mL) was added conc. HCl (0.5 mL). Thereaction mixture was stirred at 70° C. for 7 h, cooled to roomtemperature and centrifuged. The precipitate was rinsed with water (1mL×2) and dried in vacuo to afford Example 10 as a white solid (13 mg,61%).

Data:

¹H NMR (CD₃OD, 500 MHz): δ 8.09 (d, J=8.0 Hz, 2H), 8.01 (d, J=9.0 Hz,1H), 7.38 (d, J=8.0 Hz, 2H), 7.01 (dd, J=9.0, 2.0 Hz, 1H), 6.93 (d,J=2.0 Hz, 1H) ppm. MS (ESI): m/z 351.0 [M+1]⁺.

Example 11 4-(7-hydroxy-2-(methylthio)-4-oxo-4H-chromen-3-yl)benzoicacid

Synthesis:

Step 1: Synthesis of methyl4-(7-methoxy-2-(methylthio)-4-oxo-4H-chromen-3-yl)benzoate

A stirred mixture of Intermediate B (100 mg, 0.33 mmol) and carbondisulfide (127 mg, 1.67 mmol) in DMF (5.0 mL) was cooled to 0° C. andNaH (24 mg, 1.0 mmol) was added. The reaction mixture was stirred at 0°C. for 30 min. Then MeI (94 mg, 0.67 mmol) was added and the reactionmixture was stirred at room temperature for 3 h. The solution wasdiluted with water (10 mL), extracted with DCM (50 mL×3). The organiclayers were dried over Na₂SO₄, concentrated and purified by silica gelcolumn chromatography (PE:EA=3:1) to afford product (36 mg (not pure),30%). MS (ESI): m/z 357.0 [M+1]⁺.

Step 2: Synthesis of4-(7-hydroxy-2-(methylthio)-4-oxo-4H-chromen-3-yl)benzoic acid (Example11)

Methyl 4-(7-methoxy-2-(methylthio)-4-oxo-4H-chromen-3-yl)benzoate (36mg, 0.101 mmol) in dry dichloromethane (5.0 mL) was cooled to 0° C.under nitrogen and BBr₃ in DCM (1.0 M, 0.2 mL, 0.202 mmol) was addedrapidly. Then the mixture was stirred at room temperature overnight. Thereaction was quenched with water and concentrated under reducedpressure. The residue was purified by prep-HPLC to afford Example 11 asan off-white solid (8.2 mg, 25%).

Data:

¹H NMR (MeOH-d₄, 500 MHz, TMS): δ 8.10 (d, J=10.0 Hz, 2H), 8.01 (d,J=8.0 Hz, 1H), 7.45 (d, J=8.0 Hz, 2H), 6.97 (d, J=8.0 Hz, 2H), 2.64 (s,3H); MS (ESI): m/z 329.0 [M+1]⁺.

Example 124-(6-chloro-7-hydroxy-4-oxo-2-(trifluoromethyl)-4H-chromen-3-yl)benzoicacid

Synthesis:

Prepared following General Scheme 1, starting from Intermediate C andTFAA in step 1 using condition a) (where reaction was stirred at roomtemperature for 2 h, then heated at 40° C. for 4 h, and was used withoutpurification). Step 2 followed c) conditions (with purification byprep-HPLC). See Example 10 for detailed example.

Data:

¹H NMR (DMSO-d₆, 500 MHz, TMS): δ 13.10 (brs, 1H), 12.25 (brs, 1H), 8.01(d, J=8.5 Hz, 2H), 8.00 (s, 1H), 7.42 (d, J=8.0 Hz, 2H), 7.16 (s, 1H);MS (ESI): m/z 384.9 [M+1]⁺.

Example 134-(6-fluoro-7-hydroxy-4-oxo-2-(trifluoromethyl)-4H-chromen-3-yl)benzoicacid

Prepared following General Scheme 1, starting from Intermediate D andTFAA in step 1 using condition a). Step 2 followed c) conditions (withpurification by prep-HPLC). See Example 10 for detailed example.

Data:

¹H NMR (MeOH-d₄, 500 MHz, TMS): 8.11 (d, J=7.5 Hz, 2H), 7.77 (d, J=10.5Hz, 1H), 7.41 (d, J=7.5 Hz, 2H); MS (ESI): m/z 369.0 [M+1]⁺.

Example 142-fluoro-4-(7-hydroxy-4-oxo-2-(trifluoromethyl)-4H-chromen-3-yl)benzoicacid

Synthesis:

Prepared following General Scheme 1, starting from Intermediate E andTFAA in step 1 using condition a) (where crude was used withoutpurification). Step 2 followed c) conditions (with aqueous workup,followed by purification by recrystallization from PE:DCM=1:1). SeeExample 10 for detailed example.

Data:

¹H NMR (MeOH-d₄, 500 MHz, TMS): 8.05˜8.01 (m, 2H), 7.22 (d, J=2.0 Hz,1H), 7.20 (s, 1H), 7.04 (dd, J=2.0 Hz, 9.0 Hz, 1H), 6.96 (d, J=2.0 Hz,1H); MS (ESI): m/z 369.0 [M+1]⁺.

Example 153-chloro-4-(7-hydroxy-4-oxo-2-(trifluoromethyl)-4H-chromen-3-yl)benzoicacid

Synthesis:

Prepared following General Scheme 1, starting from Intermediate F andTFAA in step 1 using condition a) (where an aqueous workup wasperformed, and crude was used without purification). Step 2 followed c)conditions (with purification by prep-HPLC). See Example 10 for detailedexample.

Data:

¹H NMR (DMSO-d₆, 500 MHz, TMS): δ 13.49 (brs, 1H), 11.28 (brs, 1H), 8.04(d, J=1.0 Hz, 1H), 7.95˜7.98 (m, 2H), 7.58 (d, J=8.0 Hz, 1H), 7.06 (dd,J=2.0, 8.5 Hz, 1H), 7.02 (d, J=2.0 Hz, 1H); MS (ESI): m/z 385.0 [M+1]⁺.

Example 164-(2-(1,1-difluoroethyl)-7-hydroxy-4-oxo-4H-chromen-3-yl)benzoic acid

Synthesis:

Step 1

To a solution of 2,2-difluoropropanoic acid (330 mg, 2.97 mmol) in DCM(5 mL) was added P₂O₅ (3.3 g, 29.7 mmol) at room temperature and themixture was stirred for 2 days to give 2,2-difluoropropanoic anhydride.To a suspension of Intermediate A-2a (170 mg, 0.59 mmol) in TEA (5 mL)was added dropwise the 2,2-difluoropropanoic anhydride in DCM at 0˜10°C. The mixture was stirred at room temperature for 2 h. The volatileswere evaporated and the residue was purified by prep-TLC (PE:EA=1:1) toafford methyl4-(2-(1,1-difluoroethyl)-7-hydroxy-4-oxo-4H-chromen-3-yl)benzoate asbrown oil (45 mg, 21%).

Step 2

followed c) conditions (with purification by recrystallization fromDCM). See Example 10 for detailed example.

Data:

¹H NMR (MeOH-d₄, 500 MHz, TMS): δ 8.07 (d, J=8.5 Hz, 2H), 8.01 (d, J=8.5Hz, 1H), 7.37 (d, J=8.5 Hz, 1H), 7.01 (dd, J=2.0, 8.5 Hz, 1H), 6.96 (d,J=2.0 Hz, 1H), 1.95 (t, J_(F—H)=18.5 Hz, 3H); MS (ESI): m/z 347.0[M+1]⁺.

Example 174-(7-hydroxy-6-methoxy-4-oxo-2-(trifluoromethyl)-4H-chromen-3-yl)benzoicacid

Synthesis:

Prepared following General Scheme 1, starting from Intermediate G andTFAA in step 1 using condition a). Step 2 followed c) conditions (withpurification by prep-HPLC). See Example 10 for detailed example.

Data:

¹H NMR (MeOH-d₄, 500 MHz, TMS): δ 8.12 (d, J=8.0 Hz, 2H), 7.52 (s, 1H),7.42 (d, J=8.5 Hz, 2H), 7.04 (s, 1H), 3.98 (s, 3H); MS (ESI): m/z 381.0[M+1]⁺.

Example 182-chloro-4-(7-hydroxy-4-oxo-2-(trifluoromethyl)-4H-chromen-3-yl)benzoicacid

Synthesis:

Prepared following General Scheme 1, starting from Intermediate H andTFAA in step 1 using condition a). Step 2 followed c) conditions (withpurification by recrystallization from DCM). See Example 10 for detailedexample.

Data:

¹H NMR (MeOH-d₄, 500 MHz, TMS): 8.15 (d, J=1.5 Hz, 1H), 8.06˜8.04 (m,2H), 7.47 (d, J=8.5 Hz, 1H), 7.06 (dd, J=2.5, 9.0 Hz, 1H), 6.99 (d,J=2.0 Hz, 1H); MS (ESI): m/z 385.0 [M+1]⁺.

Example 194-(2-(1,1-difluoropropyl)-7-hydroxy-4-oxo-4H-chromen-3-yl)benzoic acid

Synthesis:

Step 1

A stirred solution of 2,2-difluorobutanoic acid (500 mg, 4.032 mmol) inCH₂Cl₂ (30 mL) was treated with P₂O₅ (5.73 g, 40.32 mmol) at roomtemperature. The reaction mixture was stored at 5° C. for 2 days. Theclear layer was added into a solution of Intermediate A-2 (300 mg, 1.049mmol) in TEA (0.4 mL, 2.77 mmol); the solid was washed with CH₂Cl₂ (1mL×3) and the combined DCM solutions were also added into the solutionof Intermediate A-2. The reaction was stirred at room temperature for 2h. The volatiles were removed under reduced pressure. The residue wastaken up in dilute HCl solution and extracted with EA. The extracts wereconcentrated to afford 300 mg of orange solid, which was purified bysilica gel column (PE:EA=10:1 to 4:1) to afford 40 mg of product (yield:10%).

Step 2

followed c) conditions (with purification by recrystallization fromDCM). See Example 10 for detailed example.

Data:

¹H NMR (MeOH-d₄, 500 MHz, TMS): δ 7.96 (d, J=8.5 Hz, 2H), 7.90 (d, J=9.0Hz, 2H), 7.24 (d, J=8.0 Hz, 2H), 6.89 (dd, J=2.0, 9.0 Hz, 1H), 6.82 (d,J=2.0 Hz, 1H), 2.17-2.09 (m, 2H), 0.91 (t, J=7.5 Hz, 3H); MS (ESI): m/z347.0 [M+1]⁺.

Example 204-(7-hydroxy-4-oxo-2-(trifluoromethyl)-4H-chromen-3-yl)-3-methoxybenzoicacid

Synthesis:

Prepared following General Scheme 1, starting from Intermediate I andTFAA in step 1 using condition a). Step 2 followed c) conditions. SeeExample 10 for detailed example.

Data:

¹H NMR (DMSO-d₆, 500 MHz, TMS): δ 13.17 (brs, 1H), 11.21 (brs, 1H), 7.93(d, J=9.0 Hz, 1H), 7.61 (dd, J=1.0, 7.5 Hz, 1H), 7.58 (s, 1H), 7.32 (d,J=8.0 Hz, 1H), 7.04 (dd, J=2.0, 8.5 Hz, 1H), 6.98 (d, J=1.5 Hz, 1H),3.77 (s, 3H); MS (ESI): m/z 381.1 [M+1]⁺.

Example 214-(6-cyano-7-hydroxy-4-oxo-2-(trifluoromethyl)-4H-chromen-3-yl)benzoicacid

Synthesis:

Step 1: Synthesis of methyl4-(6-bromo-7-methoxy-4-oxo-2-(trifluoromethyl)-4H-chromen-3-yl)benzoate

Prepared following General Scheme 1, starting from Intermediate J andTFAA using condition a) (where crude was purified by silica gel column(PE/EA=10/1 to 3/1).

Step 2: Synthesis of methyl4-(6-cyano-7-methoxy-4-oxo-2-(trifluoromethyl)-4H-chromen-3-yl)benzoate

To a solution of methyl4-(6-bromo-7-methoxy-4-oxo-2-(trifluoromethyl)-4H-chromen-3-yl)benzoate(350 mg, 0.76 mmol) in NMP (3 mL) was added CuCN (340 mg, 3.8 mmol). Themixture was stirred at 150° C. for 8 h under the protection of nitrogen.The mixture was cooled to room temperature and quenched with water (10mL) and filtered. The cake was washed with acetone (15 mL×2). Thefiltrate was concentrated in vacuo to give brown oil, which was purifiedby prep-TLC (PE/EA=5/1) to give product (125 mg, 40%) as a yellow solid.MS (ESI): 404.0 [M+1]⁺.

Step 3: Synthesis of4-(6-cyano-7-hydroxy-4-oxo-2-(trifluoromethyl)-4H-chromen-3-yl)benzoicacid

To a solution of methyl4-(6-cyano-7-methoxy-4-oxo-2-(trifluoromethyl)-4H-chromen-3-yl)benzoate(120 mg, 0.29 mmol) in DCM (3 mL) was added dropwise BBr₃ (0.35 mL, 4.5mmol) carefully at room temperature. When the addition was complete, themixture was stirred for two days. Water was added carefully and theresultant mixture was extracted with ethyl acetate (10 mL×2). Thecombined organic phase was washed with brine (5 mL), dried over Na₂SO₄and concentrated to give brown oil, which was purified by prep-HPLC toafford Example 21 (30 mg, 27.8%) as a yellow powder.

Data:

¹H NMR (MeOH-d₄, 500 MHz, TMS): δ 8.38 (s, 1H), 8.11 (d, J=8.0 Hz, 2H),7.41 (s, J=8.0 Hz, 2H), 7.09 (s, 1H); MS (ESI): m/z 376.0 [M+1]⁺.

Example 22 4-(7-hydroxy-4-oxo-4H-chromen-3-yl)benzoic acid

Synthesis:

Step 1: Synthesis of ethyl 4-(7-hydroxy-4-oxo-4H-chromen-3-yl)benzoate

To a solution of Intermediate A-1a (300 mg, 1.0 mmol) in dried DMF (8mL) was added BF₃.Et₂O (1.2 mL) carefully at 10° C. with stirring, afterthe addition was complete, the mixture was warmed to rt for 0.5 h. Thenthe reaction solution was heated to 60° C. and MsCl (2 mL in 4 mL ofDMF) was added. Then the reaction solution was heated to 95° C. for 5 h.The reaction mixture was cooled to room temperature and poured intoice-water (30 mL) and extracted with EA (20 mL×5). The organic phase waswashed with brine (20 mL), dried and concentrated in vacuo to give brownoil (160 mg, 51.6%). MS (ESI): m/z 311.0 [M+1]⁺.

Step 1: Synthesis of 4-(7-hydroxy-4-oxo-4H-chromen-3-yl)benzoic acid(Compound 22)

To a mixture of compound ethyl4-(7-hydroxy-4-oxo-4H-chromen-3-yl)benzoate (160 mg, 0.52 mmol) in1,4-dioxane (4 mL) was added Conc. HCl (2 mL). The reaction mixture washeaded to reflux overnight. The mixture was cooled to room temperatureand filtered. The filtered mass was washed with water (5 mL) 2 times andethanol (2 mL) and dried in vacuo to afford Example 22 as a brown solid(47.3 mg, 32.5%). ¹H NMR (MeOH-d₄, 400 MHz, TMS): δ 8.32 (s, 1H), 8.10(d, J=8 Hz, 3H), 7.71 (d, J=8.4 Hz, 2H), 6.99 (d, J=11.2 Hz, 1H), 6.91(s, 1H); MS (ESI): m/z 283.1 [M+1]⁺.

This Example is included as a comparative example. The IC₅₀ for thiscompound as determined by the method described in Example 24 was 135 nM.

Example 23 Synthesis of Intermediates

The syntheses of the intermediates referenced in the above examples aredescribed here. Exemplary detailed methods are described in Methods 1-3shown below in the description of Intermediate A-1a and IntermediateA-2a.

General Structure of Intermediates

Synthesis of Intermediate A-1a: ethyl4-(2-(2,4-dihydroxyphenyl)-2-oxoethyl)benzoate) and Intermediate A-2a:methyl 4-(2-(2,4-dihydroxyphenyl)-2-oxoethyl)benzoate)

The Intermediate A-1a was first synthesized by Method 1, then laterprepared by the shorter procedure described in Method 2. Method 3describes the methyl ester Intermediate A-2a in another procedure.

Method 1 Synthesis of ethyl4-(2-(2,4-dihydroxyphenyl)-2-oxoethyl)benzoate

Step 1: Synthesis of ethyl 2-(4-acetylphenyl)acetate (A-2)

To a solution of ethyl 2-phenylacetate (A-1) (50.9 g, 305 mmol) in CS₂(220 mL) was added AlCl₃ (93.6 g, 702 mmol) at 0˜10° C. over 10 min.After addition, acetyl chloride (30.5 mL, 427 mmol) was added at 0˜10°C. over 10 min. After addition, the reaction mixture was slowly heatedto reflux overnight, then poured into ice-cooled 5N HCl solution (600mL), extracted with EA (200 mL×3). The combined organic phase was washedwith water (200 mL), sat. NaHCO₃ (200 mL) and brine (200 mL), dried overNa₂SO₄, concentrated to afford a brown oil. Recrystallized withPE/acetone (150 mL/20 mL) in refrigerator to afford A-2 as a yellowcrystal solid (13.8 g, 19%).

Step 2: Synthesis of 4-(carboxymethyl)benzoic acid (A-3)

To a solution of A-2 (10.0 g, 48.5 mmol) in THF/H₂O (v/v=1/1, 100 mL)was added NaOH (3.88 g, 97.0 mmol). The reaction solution was stirred atroom temperature overnight. THF was removed under reduced pressure, thenNaOH (17.46 g, 436.5 mmol) and water (150 mL) was added. Iodine wasadded portion wise at room temperature. After addition, the reactionmixture was stirred at room temperature for 30 min, then at 90° C. for 2h. Cooled to room temperature and filtered. The filtrate was adjusted topH=8˜9 with conc. HCl. NaHSO₃ solid was added portion wise till thecolor of reaction mixture changed from brown to yellow. Conc. HCl wasadded to pH=2˜3. The resultant precipitate was filtered, washed withwater (10 mL×2), dried in vacuo to afford A-3 as a yellow powder (4.47g, 50%).

Step 3: Synthesis of ethyl 4-(2-ethoxy-2-oxoethyl)benzoate (A-4)

To a solution of A-3 (5.0 g, 27.7 mol) in DCM (50 mL) was added oxalylchloride (12.6 g, 99.2 mmol) and one drop of DMF. The mixture wasstirred at room temperature for 2 h, and then concentrated to dryness.EtOH (100 mL) was added and the mixture was stirred at room temperaturefor 2 h, then concentrated to dryness and purified to by silica gelcolumn chromatography (PE:EA=5:1) to afford A-4 as a brown oil (4.587 g,70%).

Step 4: Synthesis of 2-(4-(ethoxycarbonyl)phenyl)acetic acid (A-5)

To a solution of A-4 (3.924 g, 16.6 mmol) in THF/H₂O/EtOH (v/v=6/6/1, 65mL) was added LiOH H₂O (731 mg, 17.4 mmol). The reaction solution wasstirred at room temperature overnight. The THF was removed under reducedpressure and the aqueous solution was acidified with 1N HCl to pH=3. Thesolid was filtered and dried in vacuo to afford A-5 as a yellow solid(3.02 g, 87%).

Step 5: Synthesis of ethyl4-(2-(2,4-dimethoxyphenyl)-2-oxoethyl)benzoate (A-6)

To a solution of A-5 (3.02 g, 14.5 mmol) in DCM (45 mL) was added oxalylchloride (7.36 g, 58.0 mmol) and one drop of DMF. The mixture wasstirred at room temperature for 2 h, and then concentrated to dryness.The residue was dissolved in DCM (100 mL), AlCl₃ (3.48 g, 26.1 mmol) and1,3-dimethoxybenzene (4.0 g, 29.0 mmol) were added at 0° C. Afteraddition, the mixture was stirred at room temperature overnight. 1N HClsolution (100 mL) was added, extracted with DCM (50 mL×3). The combinedorganic phase was washed with sat. NaHCO₃ (80 mL) and brine (50 mL),dried over Na₂SO₄, concentrated and purified by silica gel columnchromatography (PE:EA=10:1) to afford A-6 as a brown oil (3.301 g, 69%).

Step 6: Synthesis of ethyl4-(2-(2,4-dihydroxyphenyl)-2-oxoethyl)benzoate (A-7)

To a solution of A-6 (3.30 g, 10.0 mmol) in DCM (50 mL) was added BBr₃(25.05 g, 100.0 mmol) at −78° C. After addition, the mixture was warmedup to room temperature and stirred overnight. 1N HCl solution (100 mL)was added carefully. The mixture was concentrated to dryness. Theresidue was dissolved in EtOH (200 mL) concentrated to dryness again.This operation repeat twice and the residue was dissolved in EtOH (80mL). SOCl₂ (35.69 g, 300.0 mmol) was added at 10° C. The mixture wasstirred at room temperature overnight, concentrated and purified bysilica gel column chromatography (PE:EA=5:1) to afford A-7 (IntermediateA-1a) as a yellow solid (2.48 g, 82%).

Method 2 Synthesis of ethyl4-(2-(2,4-dihydroxyphenyl)-2-oxoethyl)benzoate

Step 1: Synthesis of 2-(4-(methoxycarbonyl)phenyl)acetic acid (B-2)

To a solution of 2-(4-bromophenyl)acetic acid (B−1) (91.3 g, 0.42 mol,1.0 eq) in MeOH (1.5 L) was added dry TEA (85.8 g, 0.85 mol, 2.0 eq) andPd(dppf)Cl₂ (3.43 g, 4.2 mmol, 1%). The solution was heated under CO gas(4 MPa) at 120° C. for 16 h. Then it was filtered and concentrated invacuo. The residue was dissolved in 500 mL of EA and 1 L of water. Themixture was neutralized by sat. NaHCO₃ to pH=7.5 and separated. Theinorganic phase was extracted with EA (500 mL×3) acidified with 1N HClto pH=5. Filtration and drying in vacuo afforded 62.8 g of B-2 (whitesolid, yield 76%). MS (ESI): m/z 195.1 [M+1]⁺.

Step 2: Synthesis of methyl4-(2-(2,4-dimethoxyphenyl)-2-oxoethyl)benzoate (B-3)

To a solution of B-2 (15 g, 77.3 mmol) and DMF (1 drop) in anhydrous DCM(150 mL) was added dropwise oxalyl chloride (33 mL, 386.0 mmol) at 0˜5°C. with stirring. After the addition was complete, the mixture wasstirred at room temperature for 2 h. TLC (PE/EA=3/1, quenched with MeOH)indicated that the reaction was complete, the volatiles were evaporatedand the residue was diluted with DCM (20 mL), which was used directlyfor next step.

To a suspension of aluminum trichloride (16.5 g, 123.7 mmol) inanhydrous DCM (80 mL) was added 1,3-dimethoxybenzene (21.3 g, 154.6mmol) at 5° C., followed by above acyl chloride solution. The mixturewas stirred at room temperature overnight, poured carefully into icy 1 NHCl (200 mL) and extracted with EA (150 mL×3). The combined organiclayers were washed with brine (200 mL), dried with anhydrous sodiumsulfate, filtered and concentrated to obtain brown oil, which waspurified by silica gel column (PE/EA=5/1) to afford B-3 (12 g, 49.6%) asa yellow solid. MS (ESI): m/z 315.1 [M+1]⁺.

Step 3: Synthesis of ethyl4-(2-(2,4-dihydroxyphenyl)-2-oxoethyl)benzoate (Intermediate A-1a)

To a solution of B-3 (55 g, 141.6 mmol) in DCM (600 mL) was addeddropwise BBr₃ (164 mL, 1.7 mol) at −10° C. When the addition wascomplete, the mixture was stirred at room temperature overnight andpoured into crashed ice (700 g) with stirring. The volatiles wereevaporated to afford a yellow solid, which was dried in high vacuo anddissolved in absolute ethanol (500 mL). To the solution was addeddropwise thionyl chloride (80 mL) at 0˜10° C. When the addition wascomplete, the resultant mixture was heated to reflux for 3 h. Thevolatiles were evaporated and the residue was partitioned between EA(600 mL) and saturated sodium carbonate (200 mL). The organic phase wasseparated, washed with brine (200 mL), dried with anhydrous sodiumsulfate, filtered and concentrated to afford brown slurry, which waspurified by SGC (PE/EA=3/1) to afford Intermediate A-1a (24.5 g, 58%) asa yellow solid. ¹H NMR (CDCl₃, 500 MHz, TMS): δ 12.58 (s, 1H), 8.01 (d,J=8.5 Hz, 2H), 7.69 (d, J=9.0 Hz, 1H), 7.33 (d, J=8.0 Hz, 2H), 7.05(brs, 1H), 6.41 (d, J=8.0 Hz, 1H), 6.37 (s, 1H), 4.38 (q, J=7.0 Hz, 2H),4.26 (s, 2H), 1.38 (t, J=7 Hz, 3H). MS (ESI): m/z 301.1 [M+1]⁺.

Method 3 Synthesis of methyl4-(2-(2,4-dihydroxyphenyl)-2-oxoethyl)benzoate

Synthesis of methyl 4-(2-(2,4-dihydroxyphenyl)-2-oxoethyl)benzoate(Intermediate A-2a)

To a solution of B-2 (see Method 2, step 1 for synthesis) (50 g, 257.5mmol) in BF₃-Et₂O (150 mL) was added resorcinol (28.4 g, 257.5 mmol) atroom temperature and the mixture was heated at 95° C. for 5.5 h. Themixture was cooled to room temperature and poured into icy 10% Na₂CO₃solution (600 mL) with stirring and extracted with EA (500 mL×4). Thecombined organic layers was washed with water (500 mL×3) and brine (500mL×2), dried over Na₂SO₄, filtered and concentrated. The residue waspurified by silica gel column (PE:EA=7:1 to 4:1) to afford IntermediateA-2a as a yellow solid (10.2 g, 13.8%). ¹H NMR (DMSO-d₆, 500 MHz): δ12.38 (s, OH), 10.72 (s, OH), 7.91˜7.95 (m, 3H), 7.43 (d, J=8 Hz, 1H),6.41 (d, J=8.5 Hz, 1H), 6.28 (d, J=1 Hz, 1H), 4.42 (s, 2H), 3.84 (s,3H); MS (ESI): m/z 287.1 [M+1]⁺.

Synthesis of Intermediate B methyl4-(2-(2-hydroxy-4-methoxyphenyl)-2-oxoethyl)benzoate

To a solution of methyl 4-(2-(2,4-dimethoxyphenyl)-2-oxoethyl)benzoate(B-3, see Method 2 of Intermediate A-1a) (2.5 g, 8.0 mmol) in DCM (25mL) was added BBr₃/DCM (8 mL, 8 mmol, 1 mol/L in DCM) at −78° C. Whenthe addition was complete, the mixture was allowed to warm to roomtemperature and stirred for 1 h. 1N HCl solution (10 mL) was addedcarefully. The mixture was washed with sat. Na₂CO₃ (20 mL), and brine(20 mL), dried over Na₂SO₄, filtered, concentrated to dryness. The crudewas purified by combiflash (PE:EA=10:1) to afford Intermediate B as ayellow solid (600 mg, 25%). ¹H NMR (CDCl₃, 500 MHz, TMS): δ 12.60 (s,1H) 8.01 (d, J=8.0 Hz, 2H), 7.71 (d, J=9.0 Hz, 1H), 7.34 (d, J=8.0 Hz,2H), 6.42 (d, J=9.0 Hz, 2H), 4.27 (s, 2H), 3.90 (s, 3H), 3.83 (s, 3H);MS (ESI): m/z 301.1 [M+1]⁺.

Synthesis of Intermediate C methyl4-(2-(5-chloro-2,4-dihydroxyphenyl)-2-oxoethyl)benzoate

Followed the procedure described in Method 3, Intermediate A-2a,starting with B-2 and 4-chlorobenzene-1,3-diol. Crude product was usedwithout purification. MS (ESI): m/z 321.0 [M+1]⁺.

Synthesis of Intermediate D methyl4-(2-(5-fluoro-2,4-dihydroxyphenyl)-2-oxoethyl)benzoate

Followed the procedure described in Method 3, Intermediate A-2, startingwith B-2 and 4-fluorobenzene-1,3-diol. MS (ESI): m/z 305.0 [M+1]⁺.

Synthesis of Intermediate E methyl4-(2-(2,4-dihydroxyphenyl)-2-oxoethyl)-2-fluorobenzoate Step 1:Synthesis of 1-bromo-4-(bromomethyl)-2-fluorobenzene

To a solution of 1-bromo-2-fluoro-4-methylbenzene (2.5 g, 13.3 mmol) intrifluoromethyl benzene (25 mL) was a mixture of NBS (2.35 g, 13.3 mmol)and AIBN (945 mg, 6.7 mmol) in five portions at 85° C. for 30 min. Themixture was stirred at 85° C. for 3 h. The insoluble was filtered offand the filtrate was evaporated to afford1-bromo-4-(bromomethyl)-2-fluorobenzene (2.7 g, 77%) as light yellowoil, which was used directly for next step.

Step 2: Synthesis of methyl 2-fluoro-4-(2-methoxy-2-oxoethyl)benzoate

To a solution of 1-bromo-4-(bromomethyl)-2-fluorobenzene (5.4 g, 20.4mmol) in MeOH (150 mL) was added TEA (2.8 mL, 20.4 mmol) and Pd(dppf)Cl₂(1.3 g, 2 mmol). The mixture was stirred at 60° C. under CO (0.5 MPa)for 2 h. Additional TEA (4.3 mL, 30.6 mmol) was added and the mixturewas heated to 120° C. under CO (4 MPa) for 20 h. Solvent removed invacuo and the residue was purified by Combi-Flash (80 g silica gel,start PE/EA=10:0 to 1:3 gradient, 60 mL/min, 60 min, 3.6 L total solventvolume) to afford product as a colorless oil (1.5 g, 33%). MS (ESI): m/z227.1 [M+1]⁺.

Step 3: Synthesis of 2-(3-fluoro-4-(methoxycarbonyl)phenyl)acetic acid

Followed procedure described in conversion of compound A-4 to compoundA-5 in Method 1 of Synthesis of Intermediate A-1a. MS (ESI): m/z 213.0[M+1]⁺.

Step 4: Synthesis of methyl4-(2-(2,4-dihydroxyphenyl)-2-oxoethyl)-2-fluorobenzoate (Intermediate E)

Followed procedure described in Method 3 (see Intermediate A-2a), using2-(3-fluoro-4-(methoxycarbonyl)phenyl)acetic acid where reaction wasstirred at 60° C. overnight and at 95° C. for 2 h). ¹H NMR (CDCl3-d₆,500 MHz, TMS): δ 12.47 (d, J=2.0 Hz, 1H), 7.94 (t, J=8.0 Hz, 1H), 7.69(d, J=8.5 Hz, 1H), 7.13 (d, J=8.0 Hz, 1H), 7.08 (d, J=11.5 Hz, 1H), 6.43(d, J=2.5 Hz, 1H), 6.41 (d, J=2.0 Hz, 1H), 4.26 (s, 2H), 3.94 (d, J=2.5Hz, 3H); MS (ESI): m/z 305.1 [M+1]⁺.

Synthesis of Intermediate F methyl3-chloro-4-(2-(2,4-dihydroxyphenyl)-2-oxoethyl)benzoate Step 1:Synthesis of methyl 3-chloro-4-methylbenzoate

To a solution of 3-chloro-4-methylbenzoic acid (20 g, 117 mmol) in MeOH(200 mL) was added dropwise thionyl chloride (25.6 mL, 352 mmol) at 0°C. with stirring. The mixture was stirred at room temperature for 5days. The reaction was concentrated. The residue was dissolved in EA(500 mL), washed with 10% Na₂CO₃ (250 mL×2), water (250 mL) and brine(250 mL), dried over Na₂SO₄, filtered and concentrated to afford productas a yellow solid (20.8 g, 96%).

Step 2: Synthesis of methyl 4-(bromomethyl)-3-chlorobenzoate

Followed Step 1 in the synthesis of Intermediate E using methyl3-chloro-4-methylbenzoate to give product (10 g, 78%). MS (ESI): m/z263.0 [M+1]⁺.

Step 3: Synthesis of methyl 3-chloro-4-(2-methoxy-2-oxoethyl)benzoate

To a solution of methyl 4-(bromomethyl)-3-chlorobenzoate (10 g, 37.95mmol) in MeOH (300 mL) was added TEA (4.2 mL, 30.36 mmol) andPd(dppf)Cl₂ (2.8 g, 3.8 mmol). The reaction was heated under 0.4 MPa COpressure at 60° C. for 3 h, filtered and concentrated. The residue waspurified by Combi-Flash (120 g silica gel, start PE/EA=10:0 to 5:1 bygradient, 60 mL/min, 60 min, 3.6 L total solvent volume) to affordproduct as a white solid (2.7 g, 29%). MS (ESI): m/z 243.0 [M+1]⁺.

Step 4: Synthesis of 2-(2-chloro-4-(methoxycarbonyl)phenyl)acetic acid

Followed procedure described in conversion of compound A-4 to compoundA-5 in Method 1 of Synthesis of Intermediate A-1a.

Step 5: Synthesis of methyl3-chloro-4-(2-(2,4-dihydroxyphenyl)-2-oxoethyl)benzoate (Intermediate F)

Followed procedure described in Method 3 (see Intermediate A-2a), using2-(2-chloro-4-(methoxycarbonyl)phenyl)acetic acid. ¹H NMR (DMSO-d₆, 500MHz, TMS): δ 12.09 (s, 1H), 10.72 (brs, 1H), 7.97˜7.88 (m, 3H), 7.57 (d,J=8.0 Hz, 1H), 6.44 (dd, J=2.5, 8.5 Hz, 1H), 6.30 (d, J=2.0 Hz, 1H),4.62 (s, 2H), 3.87 (s, 3H); MS (ESI): m/z 321.1 [M+1]⁺.

Synthesis of Intermediate G methyl4-(2-(2,4-dihydroxy-5-methoxyphenyl)-2-oxoethyl)benzoate Step 1:Synthesis of 4-formyl-2-methoxyphenyl acetate

To a solution of of 4-hydroxy-3-methoxybenzaldehyde (10 g, 66 mmol) in30 mL of THF was added Ac₂O (8 g, 80 mmol) and TEA (20 g, 198 mmol). Thereaction was stirred at room temperature overnight. The volatiles wereevaporated to afford 15 g of product as an oil, which was used for nextstep without further purification.

Step 2: Synthesis of 4-(formyloxy)-2-methoxyphenyl acetate

To a solution of 4-formyl-2-methoxyphenyl acetate (15 g, 77 mmol) inCH₂Cl₂ (100 mL) was added m-CPBA (31 g, 154 mmol). The solution wasrefluxed at 50° C. for 2.5 h. The solution was washed with saturatedNa₂SO₃ solution (50 mL), saturated Na₂CO₃ solution (50 mL) and brine (50mL), dried over Na₂SO₄, filtrated and concentrated in vacuo to afford 16g of crude product as a white solid, which was used for next stepwithout further purification.

Step 3: Synthesis of 4-methoxybenzene-1,3-diol

To a solution of 4-(formyloxy)-2-methoxyphenyl acetate (16 g, 76 mmol)in THF (50 mL)/H₂O (50 mL) was added LiOH (7.7 g, 184.2 mmol). Thereaction was stirred at room temperature overnight. The volatiles wereremoved under reduced pressure. The residue was extracted with EA (50mL×3). The organic phase was washed with brine (50 mL), dried overNa₂SO₄, filtrated and concentrated in vacuo to give black oil, which waspurified by silica gel column (PE:EA=3:1) to afford 4.8 g of product(40%) as a brown solid.

Step 4: Synthesis of methyl4-(2-(2,4-dihydroxy-5-methoxyphenyl)-2-oxoethyl)benzoate (IntermediateG)

Followed procedure described in Method 3 (see Intermediate A), using4-methoxybenzene-1,3-diol (where reaction was heated at 60° C.overnight). MS (ESI): m/z 317.0 [M+1]⁺.

Synthesis of Intermediate H methyl2-chloro-4-(2-(2,4-dihydroxyphenyl)-2-oxoethyl)benzoate Step 1:Synthesis of 1-bromo-4-(bromomethyl)-2-chlorobenzene

Followed Step 1 of Intermediate E using1-bromo-2-chloro-4-methylbenzene.

Step 2: Synthesis of methyl 2-chloro-4-(2-methoxy-2-oxoethyl)benzoate

Followed Step 2 of Intermediate E. MS (ESI): m/z 243.0 [M+1]⁺.

Step 3: Synthesis of 2-(3-chloro-4-(methoxycarbonyl)phenyl)acetic acid

Followed Step 4 of Intermediate F. MS (ESI): m/z 229.0 [M+1]⁺.

Step 4: Synthesis of methyl2-chloro-4-(2-(2,4-dihydroxyphenyl)-2-oxoethyl)benzoate (Intermediate H)

Followed Step 5 of Intermediate F. MS (ESI): m/z 321.7 [M+1]⁺.

Synthesis of Intermediate I methyl4-(2-(2,4-dihydroxyphenyl)-2-oxoethyl)-3-methoxybenzoate Step 1:Synthesis of methyl 3-methoxy-4-methylbenzoate

starting with 3-methoxy-4-methylbenzoic acid (see step 1 of IntermediateF). MS (ESI): m/z 181.1 [M+1]⁺.

Step 2: Synthesis of methyl 4-(bromomethyl)-3-methoxybenzoate

(see step 2 of Intermediate F). MS (ESI): m/z 259.1 [M+1]⁺.

Step 3: Synthesis of methyl 3-methoxy-4-(2-methoxy-2-oxoethyl)benzoate

(see step 3 of Intermediate F). MS (ESI): m/z 239.1 [M+1]⁺.

Step 4: Synthesis of 2-(2-methoxy-4-(methoxycarbonyl)phenyl)acetic acid

(see step 4 of Intermediate F). MS (ESI): m/z 225.1 [M+1]⁺.

Step 5: Synthesis of methyl4-(2-(2,4-dihydroxyphenyl)-2-oxoethyl)-3-methoxybenzoate

(see step 5 of Intermediate F). ¹H NMR (DMSO-d₆, 500 MHz, TMS): δ 12.33(s, 1H), 10.68 (s, 1H), 7.93 (d, J=9.0 Hz, 1H), 7.55 (dd, J=1.0, 7.0 Hz,1H), 7.48 (d, J=1.5 Hz, 1H), 7.34 (d, J=8.0 Hz, 1H), 6.41 (dd, J=2.0,9.0 Hz, 1H), 6.27 (d, J=2.5 Hz, 1H), 4.36 (s, 2H), 3.86 (s, 3H), 3.79(s, 3H); MS (ESI): m/z 317.1 [M+1]⁺.

Synthesis of Intermediate J methyl4-(2-(5-bromo-2-hydroxy-4-methoxyphenyl)-2-oxoethyl)benzoate Step 1:Synthesis of methyl4-(2-(5-bromo-2,4-dimethoxyphenyl)-2-oxoethyl)benzoate

Followed Step 2 of Method 2 (see Intermediate A) using B-2 and1-bromo-2,4-dimethoxybenzene. MS (ESI): m/z 395.0 [M+1]⁺.

Step 2: Synthesis of methyl4-(2-(5-bromo-2-hydroxy-4-methoxyphenyl)-2-oxoethyl)benzoate(Intermediate J)

To a solution of methyl4-(2-(5-bromo-2,4-dimethoxyphenyl)-2-oxoethyl)benzoate (1.1 g, 2.8 mmol)in DCM (100 mL) was added AlCl₃ (7.4 g, 56 mmol) and the mixture wasstirred at room temperature for 1 h. Then the mixture was poured intoice-water (100 mL), extracted with DCM (20 mL×4). The organic phase waswashed with brine (30 mL), dried with Na₂SO₄, filtered and the filtratewas concentrated in vacuo to give brown oil, which was purified byprep-TLC (PE/EA=3/1) to afford Intermediate J (750 mg, 71%) as a lightyellow solid. MS (ESI): m/z 379.0 [M+1]⁺.

Example 24 GSNOR Assays

Various compounds were tested in vitro for their ability to inhibitGSNOR activity. GSNOR expression and purification is described inBiochemistry 2000, 39, 10720-10729.

GSNOR Fermentation:

Pre-cultures were grown from stabs of a GSNOR glycerol stock in 2XYTmedia containing 100 ug/ml ampicillin after an overnight incubation at37° C. Cells were then added to fresh 2XYT (4 L) containing ampicillinand grown to an OD (A₆₀₀) of 0.6-0.9 at 37° C. before induction. GSNORexpression was induced with 0.1% arabinose in an overnight incubation at20° C.

GSNOR Purification:

E. coli cell paste was lysed by nitrogen cavitation and the clarifiedlysate purified by Ni affinity chromatography on an AKTA FPLC (AmershamPharmacia). The column was eluted in 20 mM Tris pH 8.0/250 mM NaCl witha 0-500 mM imidazole gradient. Eluted GSNOR fractions containing theSmt-GSNOR fusion were digested overnight with Ulp-1 at 4° C. to removethe affinity tag then re-run on the Ni column under the same conditions.GSNOR was recovered in the flowthrough fraction and for crystallographyis further purified by Q-Sepharose and Heparin flowthroughchromatography in 20 mM Tris pH 8.0, 1 mM DTT, 10 uM ZnSO₄.

GSNOR Assay:

GSNO and Enzyme/NADH Solutions are made up fresh each day. The Solutionsare filtered and allowed to warm to room temperature. GSNO Solution: 100mM NaPO4 (pH 7.4), 0.480 mM GSNO. 396 μL of GSNO Solution is added to acuvette followed by 8 μL of test compound in DMSO (or DMSO only for fullreaction control) and mixed with the pipette tip. Compounds to be testedare made up at a stock concentration of 10 mM in 100% DMSO. 2 foldserial dilutions are done in 100% DMSO. 8 μL of each dilution are addedto an assay so that the final concentration of DMSO in the assay is 1%.The concentrations of compounds tested range from 100 to 0.003 μM.Enzyme/NADH Solution: 100 mM NaPO4 (pH 7.4), 0.600 mM NADH, 1.0 μg/mLGSNO Reductase. 396 μL of the Enzyme/NADH Solution is added to thecuvette to start the reaction. The cuvette is placed in the Cary 3EUV/Visible Spectrophotometer and the change in 340 nm absorbance/min at25° C. is recorded for 3 minutes. The assays are done in triplicate foreach compound concentration. IC50's for each compound are calculatedusing the standard curve analysis in the Enzyme Kinetics Module ofSigmaPlot.

Final assay conditions: 100 mM NaPO4, pH 7.4, 0.240 mM GSNO, 0.300 mMNADH, 0.5 μg/mL GSNO Reductase and 1% DMSO. Final volume: 800μL/cuvette.

GSNOR Inhibitor Activity:

GSNOR inhibitor activity was determined and IC₅₀ values were obtainedfor the compounds described in Examples 1-21. GSNOR inhibitor compoundsExamples 1-21 had an IC₅₀ of about <5 μM. GSNOR inhibitor compoundsExamples 1, 2, 3, 5, 6, 10, 13, 14, 15, 16, 18, 19, 20 had an IC₅₀ ofabout less than 0.5 μM. GSNOR inhibitor compounds Examples 1, 10, 14,15, 16, 18, and 20 had an IC₅₀ of about less than 0.1 μM.

Example 25 Mouse Pharmacokinetic (PK) Study Experimental Model

The mouse is used to determine the pharmacokinetics of GSNOR inhibitors.This species is widely used to assess the bioavailability of compoundsby administering both oral (PO) and intravenous (IV) test articles.Efficacy of the GSNOR inhibitors can be compared by assessing plasmaexposure in male BALB/c mice either via IV or PO administration at thetimes of peak activity.

Materials and Methods IV Administration of GSNOR Inhibitors

The GSNOR inhibitor Compound 10 was reconstituted in a phosphatebuffered saline (PBS)/10% Solutol (HS 15) clear solution resulting in aconcentration of 0.2 mg/mL and administered to mice (2 mg/kg) as asingle IV dose. Animals were dosed via the lateral tail vein. Bloodsamples were collected at designated time points (0.083, 0.25, 0.5, 1,2, 4, 8, 16, 24 hours) by cardiac puncture under isoflurane anesthesia(up to 1 mL blood per animal). The blood was collected into tubescontaining Li-Heparin. The blood samples were kept on ice untilcentrifugation within approximately 30 minutes of collection. The plasmawas transferred into labeled polypropylene tubes and frozen at −70° C.until analyzed by LC/MS/MS.

PO Administration of GSNOR Inhibitors

The GSNOR inhibitor Compound 10 was reconstituted in 40% PropyleneGlycol/40% Propylene Carbonate/20% of a 5% Sucrose clear solutionresulting in a concentration of 2 mg/mL and administered to mice (10mg/kg) as a single oral dose via gavage. Blood samples were collected at0.25, 0.5, 1, 2, 4, 8, 12, 16, 20 and 24 hours post dose by cardiacpuncture under isoflurane anesthesia. The blood was collected in tubescontaining Li-Heparin. The blood samples were kept on ice untilcentrifugation within approximately 30 minutes of collection. The plasmawas transferred into labeled polypropylene tubes and frozen at −70° C.until analyzed by LC/MS/MS.

LC/MS/MS Analysis

Plasma samples at each timepoint were analyzed using a LC-MS/MS with alower limit of quantification (LLOQ) of 1 ng/mL. Plasma was analyzed todetermine the amount of the GSNOR inhibitor in each sample andregression curves were generated for each GSNOR inhibitor in therelevant matrixes.

WinNonlin analysis was used for calculating PK parameters for both theIV and PO administrations:

-   -   PK parameters for IV portion—AUC_(last); AUC_(INF); T1/2; Cl;        Vss; C_(max); MRT    -   PK parameters for PO portion—AUC_(last); AUC_(INF); T1/2;        C_(max); Cl, MRT.

In addition to the above PK parameters, calculation of bioavailability(% F) was performed.

Results

IV administration: Plasma levels of GSNOR inhibitor Compound 10 weremeasured up to 24 hours post-dose.

Oral administration: Plasma levels of Compound 10 were measured up to 24hours post dose, based on area under the curve a mean oralbioavailability was determined. Compound 10 had an oral bioavailabilityof greater than 17 percent.

Example 26 Efficacy of GSNORi in Experimental Asthma Experimental AsthmaModel:

A mouse model of ovalbumin (OVA)-induced asthma is used to screen GSNORinhibitors for efficacy against methacholine (MCh)-inducedbronchoconstriction/airway hyper-reactivity. This is a widely used andwell characterized model that presents with an acute, allergic asthmaphenotype with similarities to human asthma. Efficacy of GSNORinhibitors are assessed using a prophylactic protocol in which GSNORinhibitors are administered prior to challenge with MCh.Bronchoconstriction in response to challenge with increasing doses ofMCh is assessed using whole body plethysmography (P_(enh); Buxco). Theamount of eosinophil infiltrate into the bronchoaveolar lavage fluid(BALF) is also determined as a measure of lung inflammation. The effectof GSNOR inhibitors are compared to vehicles and to Combivent (inhaled;IH) as the positive control.

Materials and Methods Allergen Sensitization and Challenge Protocol

OVA (500 μg/ml) in PBS is mixed with equal volumes of 10% (w/v) aluminumpotassium sulfate in distilled water and incubated for 60 min. at roomtemperature after adjustment to pH 6.5 using 10 N NaOH. Aftercentrifugation at 750×g for 5 min, the OVA/alum pellet is resuspended tothe original volume in distilled water. Mice receive an intraperitoneal(IP) injection of 100 μg OVA (0.2 mL of 500 μg/mL in normal saline)complexed with alum on day 0. Mice are anesthetized by IP injection of a0.2-mL mixture of ketamine and xylazine (0.44 and 6.3 mg/mL,respectively) in normal saline and are placed on a board in the supineposition. Two hundred fifty micrograms (100 μl of a 2.5 mg/ml) of OVA(on day 8) and 125 μg (50 μl of 2.5 mg/ml) OVA (on days 15, 18, and 21)are placed on the back of the tongue of each animal.

Pulmonary Function Testing (Penh)

In vivo airway responsiveness to methacholine is measured 24 h after thelast OVA challenge in conscious, freely moving, spontaneously breathingmice with whole body plethysmography using a Buxco chamber (Wilmington,N.C.). Mice are challenged with aerosolized saline or increasing dosesof methacholine (5, 20 and 50 mg/mL) generated by an ultrasonicnebulizer for 2 min. The degree of bronchoconstriction is expressed asenhanced pause (P_(enh)), a calculated dimensionless value, whichcorrelates with the measurement of airway resistance, impedance, andintrapleural pressure in the same mouse. P_(enh) readings are taken andaveraged for 4 min. after each nebulization challenge. P_(enh) iscalculated as follows: P_(enh)=[(T_(e)/T_(r)−1)×(PEF/PIF)], where T_(e)is expiration time, T_(r) is relaxation time, PEF is peak expiratoryflow, and PIF is peak inspiratory flow×0.67 coefficient. The time forthe box pressure to change from a maximum to a user-defined percentageof the maximum represents the relaxation time. The T_(r) measurementbegins at the maximum box pressure and ends at 40%.

Eosinophil Infiltrate in BALF

After measurement of airway hyper-reactivity, the mice are exsanguinatedby cardiac puncture, and then BALF is collected from either both lungsor from the right lung after tying off the left lung at the mainstembronchus. Total BALF cells are counted from a 0.05 mL aliquot, and theremaining fluid is centrifuged at 200×g for 10 min at 4° C. Cell pelletsare resuspended in saline containing 10% BSA with smears made on glassslides. Eosinophils are stained for 5 min with 0.05% aqueous eosin and5% acetone in distilled water, rinsed with distilled water, andcounterstained with 0.07% methylene blue.

GSNOR Inhibitors and Controls

GSNOR inhibitors are reconstituted in phosphate buffered saline (PBS),pH 7.4, at concentrations ranging from 0.00005 to 3 mg/mL. GSNORinhibitors are administered to mice (10 mL/kg) as a single dose eitherintravenously (IV) or orally via gavage. Dosing is performed from 30min. to 24 h prior to MCh challenge. Effect of GSNOR inhibitors arecompared to PBS vehicle dosed in the same manner.

Combivent is used as the positive control in all studies. Combivent(Boehringer Ingelheim) is administered to the lung using the inhalerdevice supplied with the product, but adapted for administration tomice, using a pipet tip. Combivent is administered 48 h, 24 h, and 1 hprior to MCh challenge. Each puff (or dose) of Combivent provides a doseof 18 μg ipatropium bromide (IpBr) and 103 μg albuterol sulfate orapproximately 0.9 mg/kg IpBr and 5 mg/kg albuterol.

Statistical Analyses

Area under the curve values for P_(enh) across baseline, saline, andincreasing doses of MCh challenge are calculated using GraphPad Prism5.0 (San Diego, Calif.) and expressed as a percent of the respective (IVor orally administered) vehicle control. Statistical differences amongtreatment groups and the respective vehicle control group within eachstudy are calculated using one-way ANOVA, Dunnetts (JMP 8.0, SASInstitute, Cary, N.C.). A p value of <0.05 among the treatment groupsand the respective vehicle control group is considered significantlydifferent.

Example 27 Efficacy of GSNOR Inhibitors in Experimental InflammatoryBowel Disease (IBD) Experimental Model

An acute model of dextran sodium sulfate (DSS)-induced IBD in mice isused to explore efficacy of GSNOR inhibitors against this disease. AcuteDSS-induced IBD is a widely used and well characterized model thatinduces pathological changes in the colon similar to those observed inthe human disease. In this model and in human disease, epithelial cellswithin the crypts of the colon are disrupted, leading to dysfunction ofthe epithelial barrier and the ensuing tissue inflammation, edema, andulceration. GSNOR inhibitor therapy may benefit IBD by restorings-nitrosogluthathione (GSNO) levels, and thus prevent or reverse theepithelial barrier dysfunction.

Experimental IBD is induced by administration of DSS in the drinkingwater over several days. GSNOR inhibitors are administered daily viaintravenous (IV) dosing. Effect of treatment is assessed via endoscopyand histopathology using a five point scale ranging from a score=0(normal tissue) to a score=4 (ulcerative tissue damage and markedpathological changes). The effect of GSNOR inhibitors is compared tovehicle treated controls. The corticosteroid, prednisolone, is used asthe positive control in this study and is administered daily via oraldosing. Naïve mice are also assessed as a normal tissue control.

Materials and Methods

Experimental IBD is induced by administration of 3% DSS in the drinkingwater on study days 0 to 5. GSNOR inhibitors are reconstituted toconcentrations of 0.2 and 2 mg/ml in phosphate buffered saline (PBS), pH7.4. Mice are treated daily via IV administration of 0.1 ml GSNORinhibitor solution per mouse for doses of 1 and 10 mg/kg/day. GSNORinhibitor dosing is started 2 days prior to the DSS administration andcontinued through the last day of the study (days −2 to 7). PBS is usedas the vehicle control and is administered in the same manner as theGSNOR inhibitor. The corticosteroid, prednisolone, is used as thepositive control for the study, and is administered orally at a dose of3 mg/kg/day on each day (study days −2 to 7).

The effect of drug treatment is assessed on day 7 via endoscopy andhistopathology. Mice are first anesthetized with inhaled isoflurane andsubjected to endoscopy using a veterinary endoscope (Karl StorzVeterinary Endoscopy America, Inc., Goleta, Calif.). Each mouse isscored for mucosal injury using the endoscopy scoring criteria. Anendoscopy score of 0 is normal, 1 is loss of vascularity, 2 is loss ofvascularity and friability, 3 is friability and erosions, and 4 isulcerations and bleeding. Following endoscopy, mice are euthanized viaasphyxiation with inhaled carbon dioxide. Colon sections are thenformalin-fixed, paraffin-embedded, sectioned, and stained withhematoxylin-eosin. Colon sections are examined via light microscopy andscored in a blinded fashion by a board certified veterinary pathologistwith particular expertise in GI pathology. Pathological changes to theepithelium, connective tissue, and submucosa are scored based oninflammation, edema, and necrosis, and a score of 0 is normal, 1 isminimal, 2 is mild, 3 is moderate, and 4 is marked.

Example 28 Efficacy of GSNOR Inhibitors in Experimental ChronicObstructive Pulmonary Disease (COPD) Experimental COPD Model

An acute model of elastase-induced COPD in mice is used to exploreefficacy of GSNOR inhibitors against this disease. Elastase-induced COPDis a widely used and well characterized model that induces pathologicalchanges in the lung similar to those observed in the human disease. Inthis model and in human disease, airway obstruction, pulmonaryinflammation, and airspace enlargement are evident. GSNOR inhibitortherapy may benefit COPD through the bronchodilatory andanti-inflammatory actions of these compounds.

Experimental COPD is induced by administration of the elastases, papainand porcine pancreatic elastase (PPE), into the lung over several days.GSNOR inhibitors are administered daily via oral dosing. Efficacy isdetermined by assessing the ability of GSNOR inhibitors to attenuatebronchoconstriction in response to methacholine (MCh) aerosol challenge,decrease pulmonary inflammation, and reduce airspace enlargement in theaveoli. The effect of GSNOR inhibitors are compared to vehicle treatedcontrols. A combination of daily oral SP CXC receptor 2/receptor 1 (SPCXCR2/1) antagonist, which blocks recruitment of neutrophils andmonocytes, and inhaled Flovent (fluticasone; corticosteroid), is used asthe positive control in this study.

Materials and Methods

Experimental COPD is induced by administration of 80 μg papain and 20U/mg PPE per mouse per day via intra-tracheal (IT) instillation on studydays 0 to 7. GSNOR inhibitor is reconstituted to concentrations of 0.01,0.1, and 1 mg/ml in phosphate buffered saline (PBS), pH 7.4. Mice aretreated daily via oral administration (gavage) of 0.1 ml GSNORi solutionper mouse for doses of 0.1, 1, and 10 mg/kg/day. PBS is used as thevehicle control and is administered via daily oral dosing. The smallmolecule antagonist SP CXCR2/R1 (Schering-Plough/Merck), which blocksreceptors to cytokine chemoattractants for neutrophil and monocyterecruitment, is used in combination with the corticosteroid, Flovent(Glaxo), as the positive control for the study. SP CXCR2/R1 is dosedorally at 50 mg/kg/day. Flovent is dosed via inhalation at 220μg/mouse/day. One group of mice is treated with GSNOR inhibitor, vehiclecontrol, or positive control for 7 days (study days 8 to 14), while asecond group of mice is treated with GSNOR inhibitor, vehicle control,or positive control for 14 days (study days 8 to 21).

The effect of drug treatment is assessed 7 and 14 days post-treatment bymeasuring attenuation of methacholine-induced bronchoconstriction(bronchodilatory effect), attenuation of pulmonary inflammation, andreduction of airspace enlargement in the alveoli (14 day post-treatmentonly).

Bronchodilatory Effect

In vivo airway responsiveness to methacholine is measured in conscious,freely moving, spontaneously breathing mice with whole bodyplethysmography using a Buxco chamber (Wilmington, N.C.). Mice arechallenged with aerosolized saline or increasing doses of methacholine(5, 20, and 50 mg/ml) generated by an ultrasonic nebulizer for 2 min.The degree of bronchoconstriction is expressed as enhanced pause (Penh),a calculated dimensionless value, which correlated with the measurementof airway resistance, impedance, and intrapleural pressure in the samemouse. Penh readings are taken and averaged for 4 min after eachnebulization challenge. Penh is calculated as follows:Penh=[(T_(e)/T_(r)−1)×(PEF/PIF)], where T_(e) is expiration time, T_(r)is relaxation time, PEF is peak expiratory flow, and PIF is peakinspiratory flow×0.67 coefficient. The time for the box pressure tochange from a maximum to a user-defined percentage of the maximumrepresented the relaxation time. The T_(r) measurement began at themaximum box pressure and ended at 40%.

Anti-Inflammatory Effect

After measurement of airway hyper-reactivity, the mice areexsanguination by cardiac puncture, and then bronchoalveolar lavagefluid (BALF) is collected from the right lung after tying off the leftlung at the mainstem bronchus. Total BALF cells are counted, and theremaining fluid is centrifuged at 200×g for 10 min. at 4° C. Cellpellets are resuspended in saline containing 10% bovine serum albumin(BSA) and smears are made on glass slides using cytospin. Cells arestained with Diff-Quik for white blood cell (WBC) differential countsvia light microscopy. Epithelial cells are counted and subtracted fromthe total number of cells. The proportions of eosinophils, macrophages,neutrophils, and lymphocytes are counted using standard morphologicalcriteria and expressed as a percentage of the total number of whiteblood cells (WBCs).

The ability of treatment to reduce levels of neutrophil and monocytechemoattractants in the BALF are also assessed as additional parametersof anti-inflammatory effect. KC (keratinocyte chemoattractant), alsoknown as GROα (growth-related oncogene alpha), and JE (MCP-1, monocytechemoattractant protein), chemokines for neutrophil and monocyterecruitment, respectively, are measured using immunoassay.

Reduction of Airspace Enlargement

Both lungs are inflated under constant positive pressure at 25 cm waterpressure with 10% buffered formaldehyde and then perfused-fixed. Thefixed lungs are embedded in paraffin, stained with hematoxylin andeosin, and examined via light microscopy. Airspace enlargement isquantified morphologically by calculating the mean linear intercept (Lm)and average equivalent diameter of alveoli (D2).

It will be apparent to those skilled in the art that variousmodifications and variations can be made in the methods and compositionsof the present invention without departing from the spirit or scope ofthe invention.

1. A method of treatment of pulmonary disorders which comprisesadministering to a patient in need thereof a therapeutically effectiveamount of a compound of Formula (I) or a pharmaceutically acceptablesalt thereof

wherein R₁ is selected from the group consisting of CF₃, CF₂H, CF₂CH₃,CF₂CH₂CH₃, isopropyl, isobutyl, cyclopentyl, CH₂OCH₃, SCH₃, benzyl,thiophen-2-yl, and thiophen-3-yl; R₂ is selected from H, F, Cl, methoxy,and cyano; and R₃ is selected from H, F, Cl, and methoxy.
 2. The methodof claim 1 wherein R₁ is selected from the group consisting of CF₃,CF₂H, and CF₂CH₃; and R₂ is hydrogen.
 3. The method of claim 1 whereinR₁ is selected from the group consisting of CF₃, isopropyl, andisobutyl; and R₂ and R₃ are both hydrogen.
 4. The method of claim 1wherein R₁ is selected from the group consisting of CF₃, isopropyl,isobutyl, CF₂H, CF₂CH₃, and CF₂CH₂CH₃; and R₂ and R₃ are both hydrogen.5. The method of claim 1 wherein the compound of Formula (I) or apharmaceutically acceptable salt thereof is selected from the groupconsisting of4-(2-(difluoromethyl)-7-hydroxy-4-oxo-4H-chromen-3-yl)benzoic acid;4-(7-hydroxy-2-(methoxymethyl)-4-oxo-4H-chromen-3-yl)benzoic acid;4-(7-hydroxy-2-isopropyl-4-oxo-4H-chromen-3-yl)benzoic acid;4-(2-cyclopentyl-7-hydroxy-4-oxo-4H-chromen-3-yl)benzoic acid;4-(2-benzyl-7-hydroxy-4-oxo-4H-chromen-3-yl)benzoic acid;4-(7-hydroxy-4-oxo-2-(thiophen-2-yl)-4H-chromen-3-yl)benzoic acid;4-(7-hydroxy-4-oxo-2-(thiophen-3-yl)-4H-chromen-3-yl)benzoic acid;4-(7-hydroxy-2-isobutyl-4-oxo-4H-chromen-3-yl)benzoic acid;4-(7-hydroxy-4-oxo-2-(trifluoromethyl)-4H-chromen-3-yl)benzoic acid;4-(7-hydroxy-2-(methylthio)-4-oxo-4H-chromen-3-yl)benzoic acid;4-(6-chloro-7-hydroxy-4-oxo-2-(trifluoromethyl)-4H-chromen-3-yl)benzoicacid;4-(6-fluoro-7-hydroxy-4-oxo-2-(trifluoromethyl)-4H-chromen-3-yl)benzoicacid;2-fluoro-4-(7-hydroxy-4-oxo-2-(trifluoromethyl)-4H-chromen-3-yl)benzoicacid;3-chloro-4-(7-hydroxy-4-oxo-2-(trifluoromethyl)-4H-chromen-3-yl)benzoicacid; 4-(2-(1,1-difluoroethyl)-7-hydroxy-4-oxo-4H-chromen-3-yl)benzoicacid;4-(7-hydroxy-6-methoxy-4-oxo-2-(trifluoromethyl)-4H-chromen-3-yl)benzoicacid;2-chloro-4-(7-hydroxy-4-oxo-2-(trifluoromethyl)-4H-chromen-3-yl)benzoicacid; 4-(2-(1,1-difluoropropyl)-7-hydroxy-4-oxo-4H-chromen-3-yl)benzoicacid;4-(7-hydroxy-4-oxo-2-(trifluoromethyl)-4H-chromen-3-yl)-3-methoxybenzoicacid; and4-(6-cyano-7-hydroxy-4-oxo-2-(trifluoromethyl)-4H-chromen-3-yl)benzoicacid.
 6. The method of claim 1 comprising administering to a patient inneed thereof a therapeutically effective amount of a compound of Formula(I) or a pharmaceutically acceptable salt thereof together with apharmaceutically accepted carrier or excipient.
 7. A method of treatmentof inflammatory disorders which comprises administering to a patient inneed thereof a therapeutically effective amount of a compound of Formula(I) or a pharmaceutically acceptable salt thereof

wherein R₁ is selected from the group consisting of CF₃, CF₂H, CF₂CH₃,CF₂CH₂CH₃, isopropyl, isobutyl, cyclopentyl, CH₂OCH₃, SCH₃, benzyl,thiophen-2-yl, and thiophen-3-yl; R₂ is selected from H, F, Cl, methoxy,and cyano; and R₃ is selected from H, F, Cl, and methoxy.
 8. The methodof claim 7 wherein R₁ is selected from the group consisting of CF₃,CF₂H, and CF₂CH₃; and R₂ is hydrogen.
 9. The method of claim 7 whereinR₁ is selected from the group consisting of CF₃, isopropyl, andisobutyl; and R₂ and R₃ are both hydrogen.
 10. The method of claim 7wherein R₁ is selected from the group consisting of CF₃, isopropyl,isobutyl, CF₂H, CF₂CH₃, and CF₂CH₂CH₃; and R₂ and R₃ are both hydrogen.11. The method of claim 7 wherein the compound of Formula (I) or apharmaceutically acceptable salt thereof is selected from the groupconsisting of4-(2-(difluoromethyl)-7-hydroxy-4-oxo-4H-chromen-3-yl)benzoic acid;4-(7-hydroxy-2-(methoxymethyl)-4-oxo-4H-chromen-3-yl)benzoic acid;4-(7-hydroxy-2-isopropyl-4-oxo-4H-chromen-3-yl)benzoic acid;4-(2-cyclopentyl-7-hydroxy-4-oxo-4H-chromen-3-yl)benzoic acid;4-(2-benzyl-7-hydroxy-4-oxo-4H-chromen-3-yl)benzoic acid;4-(7-hydroxy-4-oxo-2-(thiophen-2-yl)-4H-chromen-3-yl)benzoic acid;4-(7-hydroxy-4-oxo-2-(thiophen-3-yl)-4H-chromen-3-yl)benzoic acid;4-(7-hydroxy-2-isobutyl-4-oxo-4H-chromen-3-yl)benzoic acid;4-(7-hydroxy-4-oxo-2-(trifluoromethyl)-4H-chromen-3-yl)benzoic acid;4-(7-hydroxy-2-(methylthio)-4-oxo-4H-chromen-3-yl)benzoic acid;4-(6-chloro-7-hydroxy-4-oxo-2-(trifluoromethyl)-4H-chromen-3-yl)benzoicacid;4-(6-fluoro-7-hydroxy-4-oxo-2-(trifluoromethyl)-4H-chromen-3-yl)benzoicacid;2-fluoro-4-(7-hydroxy-4-oxo-2-(trifluoromethyl)-4H-chromen-3-yl)benzoicacid;3-chloro-4-(7-hydroxy-4-oxo-2-(trifluoromethyl)-4H-chromen-3-yl)benzoicacid; 4-(2-(1,1-difluoroethyl)-7-hydroxy-4-oxo-4H-chromen-3-yl)benzoicacid;4-(7-hydroxy-6-methoxy-4-oxo-2-(trifluoromethyl)-4H-chromen-3-yl)benzoicacid;2-chloro-4-(7-hydroxy-4-oxo-2-(trifluoromethyl)-4H-chromen-3-yl)benzoicacid; 4-(2-(1,1-difluoropropyl)-7-hydroxy-4-oxo-4H-chromen-3-yl)benzoicacid;4-(7-hydroxy-4-oxo-2-(trifluoromethyl)-4H-chromen-3-yl)-3-methoxybenzoicacid; and4-(6-cyano-7-hydroxy-4-oxo-2-(trifluoromethyl)-4H-chromen-3-yl)benzoicacid.
 12. The method of claim 7 comprising administering to a patient inneed thereof a therapeutically effective amount of a compound of Formula(I) or a pharmaceutically acceptable salt thereof together with apharmaceutically accepted carrier or excipient.